Structured Review

Millipore filipin
Caveolin-mediated endocytic mechanisms are implicated in internalization of rMGA_0676. (A) Internalization of rMGA_0676 was blocked by caveolin-mediated endocytic inhibitor <t>filipin.</t> DF-1 cells were treated with filipin followed by rMGA_0676 (40 μg/ml) for 24 h at 37°C to detect the inhibition of rMGA_0676 internalization. The samples underwent IFA and were examined with a laser confocal scanning microscope. Cellular F-actin was stained with Alexa Fluor 555-conjugated phalloidin. The nuclei were counterstained with DAPI (blue). The rMGA_0676 was hybridized with mouse anti-His monoclonal antibody and labeled with FITC-conjugated goat anti mouse IgG antibody green fluorescence (GF). The normal DF-1 cells as the negative control (Con), cells were treated with Cholera toxin-FITC as the caveolin-mediated endocytic positive control (Cholera toxin). (B) The mean value of GF was used to quantify the positive staining for rMGA_0676 in filipin treated DF-1 cells (A) , the data were analyzed using SPSS software, and the graph was made using GraphPad Prism 5.0. “ ** ”represented statistically significant differences ( P
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Images

1) Product Images from "Mechanism of Apoptosis Induction by Mycoplasmal Nuclease MGA_0676 in Chicken Embryo Fibroblasts"

Article Title: Mechanism of Apoptosis Induction by Mycoplasmal Nuclease MGA_0676 in Chicken Embryo Fibroblasts

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00105

Caveolin-mediated endocytic mechanisms are implicated in internalization of rMGA_0676. (A) Internalization of rMGA_0676 was blocked by caveolin-mediated endocytic inhibitor filipin. DF-1 cells were treated with filipin followed by rMGA_0676 (40 μg/ml) for 24 h at 37°C to detect the inhibition of rMGA_0676 internalization. The samples underwent IFA and were examined with a laser confocal scanning microscope. Cellular F-actin was stained with Alexa Fluor 555-conjugated phalloidin. The nuclei were counterstained with DAPI (blue). The rMGA_0676 was hybridized with mouse anti-His monoclonal antibody and labeled with FITC-conjugated goat anti mouse IgG antibody green fluorescence (GF). The normal DF-1 cells as the negative control (Con), cells were treated with Cholera toxin-FITC as the caveolin-mediated endocytic positive control (Cholera toxin). (B) The mean value of GF was used to quantify the positive staining for rMGA_0676 in filipin treated DF-1 cells (A) , the data were analyzed using SPSS software, and the graph was made using GraphPad Prism 5.0. “ ** ”represented statistically significant differences ( P
Figure Legend Snippet: Caveolin-mediated endocytic mechanisms are implicated in internalization of rMGA_0676. (A) Internalization of rMGA_0676 was blocked by caveolin-mediated endocytic inhibitor filipin. DF-1 cells were treated with filipin followed by rMGA_0676 (40 μg/ml) for 24 h at 37°C to detect the inhibition of rMGA_0676 internalization. The samples underwent IFA and were examined with a laser confocal scanning microscope. Cellular F-actin was stained with Alexa Fluor 555-conjugated phalloidin. The nuclei were counterstained with DAPI (blue). The rMGA_0676 was hybridized with mouse anti-His monoclonal antibody and labeled with FITC-conjugated goat anti mouse IgG antibody green fluorescence (GF). The normal DF-1 cells as the negative control (Con), cells were treated with Cholera toxin-FITC as the caveolin-mediated endocytic positive control (Cholera toxin). (B) The mean value of GF was used to quantify the positive staining for rMGA_0676 in filipin treated DF-1 cells (A) , the data were analyzed using SPSS software, and the graph was made using GraphPad Prism 5.0. “ ** ”represented statistically significant differences ( P

Techniques Used: Inhibition, Immunofluorescence, Microscopy, Staining, Labeling, Fluorescence, Negative Control, Positive Control, Software

2) Product Images from "Lysosomal Accumulation of SCMAS (Subunit c of Mitochondrial ATP Synthase) in Neurons of the Mouse Model of Mucopolysaccharidosis III B"

Article Title: Lysosomal Accumulation of SCMAS (Subunit c of Mitochondrial ATP Synthase) in Neurons of the Mouse Model of Mucopolysaccharidosis III B

Journal:

doi: 10.1016/j.ymgme.2006.11.006

Storage products revealed by staining and immunostaining in selected areas of the brain of 6 months-old mice. All panels show sections from the medial entorhinal cortex, except for Panels C and D which show sections from the somatosensory cortex. Panels A - D: SCMAS immunostaining and hematoxylin counterstain; A and C from MPS III B mouse, B and D, from wild type control. Panels E and F: glycosaminoglycan stained with colloidal iron in sections from MPS III B (E) and wild type control (F) mice. Panels G and H: unesterified cholesterol fluorescence upon reaction with filipin in sections from MPS III B (G) and wild type control (H) mice. Panels I -L: double immunofluorescence of SCMAS with Lamp 1, ubiquitin and GM3, as indicated on the figure; Panel M: double immunofluorescence of Tom 20 with Lamp1. The scale bars represent 100 μm for panels A-K, while the insets show cells at 10 times higher magnification for panels A-D and 4 times higher magnification for panels E and G. The scale bars for panels L and M represent 10 μm.
Figure Legend Snippet: Storage products revealed by staining and immunostaining in selected areas of the brain of 6 months-old mice. All panels show sections from the medial entorhinal cortex, except for Panels C and D which show sections from the somatosensory cortex. Panels A - D: SCMAS immunostaining and hematoxylin counterstain; A and C from MPS III B mouse, B and D, from wild type control. Panels E and F: glycosaminoglycan stained with colloidal iron in sections from MPS III B (E) and wild type control (F) mice. Panels G and H: unesterified cholesterol fluorescence upon reaction with filipin in sections from MPS III B (G) and wild type control (H) mice. Panels I -L: double immunofluorescence of SCMAS with Lamp 1, ubiquitin and GM3, as indicated on the figure; Panel M: double immunofluorescence of Tom 20 with Lamp1. The scale bars represent 100 μm for panels A-K, while the insets show cells at 10 times higher magnification for panels A-D and 4 times higher magnification for panels E and G. The scale bars for panels L and M represent 10 μm.

Techniques Used: Staining, Immunostaining, Mouse Assay, Fluorescence, Immunofluorescence

3) Product Images from "cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells"

Article Title: cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells

Journal:

doi: 10.1152/ajplung.90402.2008

Correction of cholesterol transport in CF model IB3 cells with Rp -cAMPS. A : representative image of IB3 cells costained for βarr2 and endogenous free cholesterol (filipin). B : representative images of CF model IB3 cells costained for βarr2
Figure Legend Snippet: Correction of cholesterol transport in CF model IB3 cells with Rp -cAMPS. A : representative image of IB3 cells costained for βarr2 and endogenous free cholesterol (filipin). B : representative images of CF model IB3 cells costained for βarr2

Techniques Used:

Correction of cholesterol transport in CF model cells with Rp -cAMPS. A : representative image of WT 9/HTEo- cells costained for βarr2 and endogenous free cholesterol (filipin). B : 3 representative images of CF model 9/HTEo- pCEPR cells costained
Figure Legend Snippet: Correction of cholesterol transport in CF model cells with Rp -cAMPS. A : representative image of WT 9/HTEo- cells costained for βarr2 and endogenous free cholesterol (filipin). B : 3 representative images of CF model 9/HTEo- pCEPR cells costained

Techniques Used:

Cholesterol accumulation in 9/HTEo- cells initiated by 8-bromo-cAMP (8-Br-cAMP). A : 3 representative images of endogenous free cholesterol content visualized by filipin staining in wild-type (WT) 9/HTEo- (pCEP) cells in the absence or presence of the
Figure Legend Snippet: Cholesterol accumulation in 9/HTEo- cells initiated by 8-bromo-cAMP (8-Br-cAMP). A : 3 representative images of endogenous free cholesterol content visualized by filipin staining in wild-type (WT) 9/HTEo- (pCEP) cells in the absence or presence of the

Techniques Used: Staining

β 2 -Adrenergic receptor (β2-AR) and cholesterol colocalize in CF model 9/HTEo- pCEPR cells. Immunostaining for β2-AR and cholesterol visualization with filipin in WT 9/HTEo- pCEP ( A ) and CF model pCEPR cells ( B ) is shown. In merged
Figure Legend Snippet: β 2 -Adrenergic receptor (β2-AR) and cholesterol colocalize in CF model 9/HTEo- pCEPR cells. Immunostaining for β2-AR and cholesterol visualization with filipin in WT 9/HTEo- pCEP ( A ) and CF model pCEPR cells ( B ) is shown. In merged

Techniques Used: Immunostaining

4) Product Images from "Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150Glued and late endosome positioning"

Article Title: Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150Glued and late endosome positioning

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200811005

LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (E D ). The color LUT visualizes the differences in the E D panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments ( > 10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in > 10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10 −6 ). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E . n > 50 for each condition. Bars, 10 µm.
Figure Legend Snippet: LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (E D ). The color LUT visualizes the differences in the E D panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments ( > 10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in > 10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10 −6 ). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E . n > 50 for each condition. Bars, 10 µm.

Techniques Used: Expressing, Cell Culture, Imaging, Confocal Laser Scanning Microscopy, Transfection, Staining

Chemical and genetic cholesterol manipulations and LEs. (A) Modulation of intracellular cholesterol levels. Mel JuSo cells were cultured in normal medium (FCS), cholesterol-depleted medium supplemented with lovastatin (statin), or normal medium supplemented with U-18666A, as indicated. Fixed cells were stained with filipin to detect cholesterol. A color look up table (LUT) shows fluorescence intensities. n > 100 for each condition. (B) Effects of intracellular cholesterol manipulation (control [CTRL], U-18666A, or statin treatment) on LE positioning in RILP- or ORP1L-silenced Mel JuSo cells. Cells were stained for the LE marker CD63 (red), endogenous ORP1L (green), and actin (blue). The position of the CD63-positive vesicles relative to the nucleus (radial distribution) was determined for the various conditions ( > 50 cells per condition), binned, and plotted (right). The distributions were statistically different according to the Jonckheere-Terpstra test for control siRNA (P = 1.52 × 10 −5 and 7.76 × 10 −6 for CD63 and ORP1L, respectively) only. No difference was detected for siRILP (P = 0.20 and 0.54 for CD63 and ORP1L, respectively) and for siORP1L (P = 0.29 for CD63). (C) Dynein motor activity and clustering of LEs in NPC1-silenced cells. Mel JuSo cells were transfected with siRNA for NPC1 for 72 h and, after 48 h, transfected with an expression construct for p50 dynamitin to disrupt the dynein–dynactin motor. Fixed cells were stained for CD63 and p50 dynamitin to mark the overexpressing cells. n > 100. Bars: (A and C)10 µm; (B) 20 µm.
Figure Legend Snippet: Chemical and genetic cholesterol manipulations and LEs. (A) Modulation of intracellular cholesterol levels. Mel JuSo cells were cultured in normal medium (FCS), cholesterol-depleted medium supplemented with lovastatin (statin), or normal medium supplemented with U-18666A, as indicated. Fixed cells were stained with filipin to detect cholesterol. A color look up table (LUT) shows fluorescence intensities. n > 100 for each condition. (B) Effects of intracellular cholesterol manipulation (control [CTRL], U-18666A, or statin treatment) on LE positioning in RILP- or ORP1L-silenced Mel JuSo cells. Cells were stained for the LE marker CD63 (red), endogenous ORP1L (green), and actin (blue). The position of the CD63-positive vesicles relative to the nucleus (radial distribution) was determined for the various conditions ( > 50 cells per condition), binned, and plotted (right). The distributions were statistically different according to the Jonckheere-Terpstra test for control siRNA (P = 1.52 × 10 −5 and 7.76 × 10 −6 for CD63 and ORP1L, respectively) only. No difference was detected for siRILP (P = 0.20 and 0.54 for CD63 and ORP1L, respectively) and for siORP1L (P = 0.29 for CD63). (C) Dynein motor activity and clustering of LEs in NPC1-silenced cells. Mel JuSo cells were transfected with siRNA for NPC1 for 72 h and, after 48 h, transfected with an expression construct for p50 dynamitin to disrupt the dynein–dynactin motor. Fixed cells were stained for CD63 and p50 dynamitin to mark the overexpressing cells. n > 100. Bars: (A and C)10 µm; (B) 20 µm.

Techniques Used: Cell Culture, Staining, Fluorescence, Marker, Activity Assay, Transfection, Expressing, Construct

5) Product Images from "The multivesicular body is the major internal site of prion conversion"

Article Title: The multivesicular body is the major internal site of prion conversion

Journal: Journal of Cell Science

doi: 10.1242/jcs.165472

Knocking down Hrs or Tsg101 alters PrPsc localization and decreases the level of PrPsc. (A) Hrs or Tsg101 was knocked down (KD) for the indicated times before immunostaining. The merge image shows PrPsc (red), LAMP1 (green) and EEA1 (blue). (B) Western blot of PrPsc and PrPc in lysates from control and in cells knocked down for the following proteins: Rab7a, Hrs, Tsg101. (C) Western blot of PrPsc in lysates from SMB cells depleted of Alix. (D) Western blot of PrPsc in lysates prepared from SMB cells stably expressing the indicated Rab constructs. Immunoblots were probed with antibodies against PrP and actin. (E) Filipin staining of SMB cells grown under different conditions. Control cells, cells incubated in U18666A (50 µM final concentration) for 2 days, Rab7a-knockdown cells, and Tsg101-knockdown cells were stained with filipin and anti-LAMP1 antibody. Scale bar, 10 µm. (F) Relative cholesterol levels under different conditions. Data (mean±s.d., n = 10) were normalized by setting the intensity of the background and U18666A-treated cells to 0% and 100%, respectively.
Figure Legend Snippet: Knocking down Hrs or Tsg101 alters PrPsc localization and decreases the level of PrPsc. (A) Hrs or Tsg101 was knocked down (KD) for the indicated times before immunostaining. The merge image shows PrPsc (red), LAMP1 (green) and EEA1 (blue). (B) Western blot of PrPsc and PrPc in lysates from control and in cells knocked down for the following proteins: Rab7a, Hrs, Tsg101. (C) Western blot of PrPsc in lysates from SMB cells depleted of Alix. (D) Western blot of PrPsc in lysates prepared from SMB cells stably expressing the indicated Rab constructs. Immunoblots were probed with antibodies against PrP and actin. (E) Filipin staining of SMB cells grown under different conditions. Control cells, cells incubated in U18666A (50 µM final concentration) for 2 days, Rab7a-knockdown cells, and Tsg101-knockdown cells were stained with filipin and anti-LAMP1 antibody. Scale bar, 10 µm. (F) Relative cholesterol levels under different conditions. Data (mean±s.d., n = 10) were normalized by setting the intensity of the background and U18666A-treated cells to 0% and 100%, respectively.

Techniques Used: Immunostaining, Western Blot, Stable Transfection, Expressing, Construct, Staining, Incubation, Concentration Assay

6) Product Images from "Annexin A6-Balanced Late Endosomal Cholesterol Controls Influenza A Replication and Propagation"

Article Title: Annexin A6-Balanced Late Endosomal Cholesterol Controls Influenza A Replication and Propagation

Journal: mBio

doi: 10.1128/mBio.00608-13

Pharmacological inhibition of late endosomal cholesterol egress inhibits influenza virus replication in A549 cells. (A) A549 cells transiently overexpressing GFP (green, left) or AnxA6-GFP (green, right) were treated with U18666A overnight. Cellular cholesterol was stained using filipin (blue). Bar, 20 µm. (B and C) A549 wild-type cells (B) or A549 cells transiently overexpressing GFP or AnxA6-GFP (C) were treated with U18666A overnight and then infected with FPV (MOI of 0.1 for 24 h). Progeny virus titers were determined by standard plaque assay. IAV M1 protein and AnxA6 expression levels were monitored by Western blotting. Equal protein loading was verified using STAT3 or α-tubulin, as indicated. Mean values ± SEM of at least three independent experiments were calculated and assessed for statistically significant differences using a two-tailed t test. *, P ≤ 0.05; **, P ≤ 0.01.
Figure Legend Snippet: Pharmacological inhibition of late endosomal cholesterol egress inhibits influenza virus replication in A549 cells. (A) A549 cells transiently overexpressing GFP (green, left) or AnxA6-GFP (green, right) were treated with U18666A overnight. Cellular cholesterol was stained using filipin (blue). Bar, 20 µm. (B and C) A549 wild-type cells (B) or A549 cells transiently overexpressing GFP or AnxA6-GFP (C) were treated with U18666A overnight and then infected with FPV (MOI of 0.1 for 24 h). Progeny virus titers were determined by standard plaque assay. IAV M1 protein and AnxA6 expression levels were monitored by Western blotting. Equal protein loading was verified using STAT3 or α-tubulin, as indicated. Mean values ± SEM of at least three independent experiments were calculated and assessed for statistically significant differences using a two-tailed t test. *, P ≤ 0.05; **, P ≤ 0.01.

Techniques Used: Inhibition, Staining, Infection, Plaque Assay, Expressing, Western Blot, Two Tailed Test

7) Product Images from "Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism"

Article Title: Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

Journal: Nature Communications

doi: 10.1038/ncomms11919

Cholesterol is redistributed to late endosomes/lysosomes in VPS35 KD HeLa cells. ( a ) Western blot analysis of VPS35 levels in HeLa cells transfected with scramble or VPS35 siRNA. Tubulin was used as an equal loading marker. ( b ) Representative confocal images of HeLa cells transfected with scramble or VPS35 siRNA, immunostained for LAMP1 (magenta) and VPS35 (green) and labelled with filipin (blue). Scale bar, 5 μm. ( c ) Normalized integrated densities of filipin staining in scramble (100±5%,±indicates s.e.m., n =29 cells, three experiments) and VPS35 siRNA cells (124±9%, n =28 cells, three experiments). * P
Figure Legend Snippet: Cholesterol is redistributed to late endosomes/lysosomes in VPS35 KD HeLa cells. ( a ) Western blot analysis of VPS35 levels in HeLa cells transfected with scramble or VPS35 siRNA. Tubulin was used as an equal loading marker. ( b ) Representative confocal images of HeLa cells transfected with scramble or VPS35 siRNA, immunostained for LAMP1 (magenta) and VPS35 (green) and labelled with filipin (blue). Scale bar, 5 μm. ( c ) Normalized integrated densities of filipin staining in scramble (100±5%,±indicates s.e.m., n =29 cells, three experiments) and VPS35 siRNA cells (124±9%, n =28 cells, three experiments). * P

Techniques Used: Western Blot, Transfection, Marker, Staining

Cholesterol is redistributed to late endosomes/lysosomes in Arf6 KO MEFs. ( a ) Western blot analysis of endogenous Arf6 levels in Arf6 Flox/Flox ; Cre-ER MEFs treated with DMSO ( Arf6 WT) or tamoxifen ( Arf6 KO). Tubulin was used as an equal loading marker. ( b ) Cholesterol levels measured by LC–MS were similar in Arf6 WT (19.1±0.7 molar % of total lipidome,±indicates s.e.m., n =11) and KO cells (18.5±0.5 molar % of total lipidome, n =12). NS denotes P > 0.05 in Student's t -test. ( c ) Representative confocal images of Arf6 WT and KO MEFs stained with filipin. Scale bar, 5 μm. ( d ) Normalized integrated densities of filipin staining were similar in Arf6 WT (100±8%,±indicates s.e.m., n=32 cells from three experiments) and Arf6 KO cells (98±8%, n =42 cells from four experiments). NS denotes P > 0.05 in Student's t -test. ( e ) Quantification of filipin puncta in Arf6 WT and KO cells. Left panel, number of puncta per cell in Arf6 WT (13±2 puncta per cell,±indicates s.e.m., n =39 cells from 3 experiments) and Arf6 KO cells (26±3 puncta per cell, n =29 cells from 3 experiments). ** P
Figure Legend Snippet: Cholesterol is redistributed to late endosomes/lysosomes in Arf6 KO MEFs. ( a ) Western blot analysis of endogenous Arf6 levels in Arf6 Flox/Flox ; Cre-ER MEFs treated with DMSO ( Arf6 WT) or tamoxifen ( Arf6 KO). Tubulin was used as an equal loading marker. ( b ) Cholesterol levels measured by LC–MS were similar in Arf6 WT (19.1±0.7 molar % of total lipidome,±indicates s.e.m., n =11) and KO cells (18.5±0.5 molar % of total lipidome, n =12). NS denotes P > 0.05 in Student's t -test. ( c ) Representative confocal images of Arf6 WT and KO MEFs stained with filipin. Scale bar, 5 μm. ( d ) Normalized integrated densities of filipin staining were similar in Arf6 WT (100±8%,±indicates s.e.m., n=32 cells from three experiments) and Arf6 KO cells (98±8%, n =42 cells from four experiments). NS denotes P > 0.05 in Student's t -test. ( e ) Quantification of filipin puncta in Arf6 WT and KO cells. Left panel, number of puncta per cell in Arf6 WT (13±2 puncta per cell,±indicates s.e.m., n =39 cells from 3 experiments) and Arf6 KO cells (26±3 puncta per cell, n =29 cells from 3 experiments). ** P

Techniques Used: Gene Knockout, Western Blot, Marker, Liquid Chromatography, Mass Spectrometry, Staining

Lowering PI4P levels through GFP-PIPKIγi1 or GFP-Sac2 overexpression rescues cholesterol redistribution in Arf6 KO cells. ( a ) Left, representative confocal images of Arf6 KO cells expressing the K188A mutant (top) or the wild-type (bottom) GFP-PIPKIγi1 (green) labelled with filipin (blue). Scale bar, 5 μm. Center, quantification of the absolute number and relative distribution of filipin puncta in Arf6 KO cells expressing K188A GFP-PIPKIγi1 (31±4 puncta per cell,±indicates s.e.m., n =24 cells, three experiments) or WT GFP-PIPKIγi1 (18±2 puncta per cell, n =38 cells, five experiments). ** P
Figure Legend Snippet: Lowering PI4P levels through GFP-PIPKIγi1 or GFP-Sac2 overexpression rescues cholesterol redistribution in Arf6 KO cells. ( a ) Left, representative confocal images of Arf6 KO cells expressing the K188A mutant (top) or the wild-type (bottom) GFP-PIPKIγi1 (green) labelled with filipin (blue). Scale bar, 5 μm. Center, quantification of the absolute number and relative distribution of filipin puncta in Arf6 KO cells expressing K188A GFP-PIPKIγi1 (31±4 puncta per cell,±indicates s.e.m., n =24 cells, three experiments) or WT GFP-PIPKIγi1 (18±2 puncta per cell, n =38 cells, five experiments). ** P

Techniques Used: Over Expression, Gene Knockout, Expressing, Mutagenesis

8) Product Images from "Hearing Loss and Hair Cell Death in Mice Given the Cholesterol-Chelating Agent Hydroxypropyl-?-Cyclodextrin"

Article Title: Hearing Loss and Hair Cell Death in Mice Given the Cholesterol-Chelating Agent Hydroxypropyl-?-Cyclodextrin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053280

Cholesterol levels are unchanged one week after treatment with 8,000 mg/kg HPβCD. (A) Total cholesterol in liver and cochlea of control and HPβCD treated animals (N = 3 for each). Error bars represent one standard deviation from the mean. (B) Representative images of filipin stained organ of Corti. Mid-apical turn of control and HPβCD treated animals is shown.
Figure Legend Snippet: Cholesterol levels are unchanged one week after treatment with 8,000 mg/kg HPβCD. (A) Total cholesterol in liver and cochlea of control and HPβCD treated animals (N = 3 for each). Error bars represent one standard deviation from the mean. (B) Representative images of filipin stained organ of Corti. Mid-apical turn of control and HPβCD treated animals is shown.

Techniques Used: Standard Deviation, Staining

9) Product Images from "Transcriptional profile of Paracoccidioides spp. in response to itraconazole"

Article Title: Transcriptional profile of Paracoccidioides spp. in response to itraconazole

Journal: BMC Genomics

doi: 10.1186/1471-2164-15-254

Sterol distribution in Paracoccidioides spp.. Yeast cells were fixed, stained with filipin and observed by fluorescence microscopy. Staining in the control Pb 01 (A) and Pb 18 (C) cells was diffuse with homogeneous labeling. Pb 01 (B) and Pb 18 (D) cells treated with itraconazole displayed heterogeneous fluorescence.
Figure Legend Snippet: Sterol distribution in Paracoccidioides spp.. Yeast cells were fixed, stained with filipin and observed by fluorescence microscopy. Staining in the control Pb 01 (A) and Pb 18 (C) cells was diffuse with homogeneous labeling. Pb 01 (B) and Pb 18 (D) cells treated with itraconazole displayed heterogeneous fluorescence.

Techniques Used: Staining, Fluorescence, Microscopy, Labeling

10) Product Images from "Role of Endolysosomes in Skeletal Muscle Pathology Observed in a Cholesterol-Fed Rabbit Model of Alzheimer’s Disease"

Article Title: Role of Endolysosomes in Skeletal Muscle Pathology Observed in a Cholesterol-Fed Rabbit Model of Alzheimer’s Disease

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2016.00129

Cholesterol-enriched diet increases accumulation of free cholesterol in endolysosomes and induces endolysosome enlargement. (A,B) Filipin-positive staining of free cholesterol (blue) co-distributed with EEA1-positive endosomes (red) and LAMP2-positive lysosomes (red) in muscle from cholesterol-fed rabbits. (C) In muscle fibers from control rabbits, EEA1, LAMP2, and cathepsin D staining appeared weak and diffuse, but in muscle fibers from cholesterol-fed rabbits, EEA1, LAMP2, and cathepsin D positive staining was strong forming large clumps (40X). (D) Cholesterol-enriched diet increased co-localization of EEA1 with LAMP2 in those abnormally enlarged endolysosomes. Bar = 20 μm.
Figure Legend Snippet: Cholesterol-enriched diet increases accumulation of free cholesterol in endolysosomes and induces endolysosome enlargement. (A,B) Filipin-positive staining of free cholesterol (blue) co-distributed with EEA1-positive endosomes (red) and LAMP2-positive lysosomes (red) in muscle from cholesterol-fed rabbits. (C) In muscle fibers from control rabbits, EEA1, LAMP2, and cathepsin D staining appeared weak and diffuse, but in muscle fibers from cholesterol-fed rabbits, EEA1, LAMP2, and cathepsin D positive staining was strong forming large clumps (40X). (D) Cholesterol-enriched diet increased co-localization of EEA1 with LAMP2 in those abnormally enlarged endolysosomes. Bar = 20 μm.

Techniques Used: Staining

11) Product Images from ""

Article Title:

Journal:

doi: 10.1074/jbc.M109.041152

Exosome fusion with parental cells. A , a panel of confocal laser-scanning microscopy images of a human metastatic melanoma is shown. NHS-rhodamine-labeled exosomes ( red ) were incubated with cells for 4 h followed by fixation and labeling with green fluorescent antibodies directed to Rab 5B (early endosomes), Lamp-1 (lysosomes), and Golgina (Golgi apparatus). Arrows indicate the events of colocalization ( yellow ) in all samples, with the exception of Golgi compartment. Images in the right column represent magnification of the images on the left column. Bars : left panels , 10 μm; right panels , 4 μm. B , R18-exoMel1 were left untreated or mixed with 1 × 106 Mel1 cells. Note that a fluorescence dequenching ( FD ) curve was observed only after the addition of the cells. Tx 100 , Triton X-100. C , R18-exoMel1 were left untreated or pretreated with 0.5% PAF before the addition to Mel1 cells, and fusion activity was tested. A representative fluorescence dequenching curve is shown. Inset , statistical analysis obtained by 20 min of kinetic experiments is shown. Values are the means ± S.D. a = p < 0.05 versus control ( n = 3). D , Mel1 cells (1 × 106 ) were left untreated or treated with filipin, then subjected to a fusion test with R18-exoMel1. A representative fluorescence dequenching curve is shown. Inset , statistical analysis obtained by 30 min kinetic experiments is shown. Values are the means ± S.D. a = p < 0.01 versus control ( n = 3). E , R18-exoMel1ac and R18-exoMel1 (10 μg) were mixed with parental cells at the corresponding pH, and fusion was monitored. A representative fluorescence dequenching curve is shown. Statistical analysis on 5-, 20-, and 30-min kinetic experiments is represented. Values are the means ± S.D. a , p < 0.05 ( n = 3). F , streptavidin blotting is shown. 20 μg of biotinylated exosomes were incubated with parental cells (1 × 106 ) at the corresponding pH ( lane 1 , exoMel1 on Mel1; lane 2 , exoMel1ac on Mel1ac ) or with filipin-treated cells ( lane 3 , exoMel1 on Mel1; lane 4 , exoMel1ac on Mel1ac ) for 1 h at 37 °C. Immunoblotting of nucleolin protein expression represents a control for protein equal loading. A representative Western blot of three independent experiments is shown. Numbers expressed in arbitrary units ( a.u .) represent streptavidin densitometry analysis.
Figure Legend Snippet: Exosome fusion with parental cells. A , a panel of confocal laser-scanning microscopy images of a human metastatic melanoma is shown. NHS-rhodamine-labeled exosomes ( red ) were incubated with cells for 4 h followed by fixation and labeling with green fluorescent antibodies directed to Rab 5B (early endosomes), Lamp-1 (lysosomes), and Golgina (Golgi apparatus). Arrows indicate the events of colocalization ( yellow ) in all samples, with the exception of Golgi compartment. Images in the right column represent magnification of the images on the left column. Bars : left panels , 10 μm; right panels , 4 μm. B , R18-exoMel1 were left untreated or mixed with 1 × 106 Mel1 cells. Note that a fluorescence dequenching ( FD ) curve was observed only after the addition of the cells. Tx 100 , Triton X-100. C , R18-exoMel1 were left untreated or pretreated with 0.5% PAF before the addition to Mel1 cells, and fusion activity was tested. A representative fluorescence dequenching curve is shown. Inset , statistical analysis obtained by 20 min of kinetic experiments is shown. Values are the means ± S.D. a = p < 0.05 versus control ( n = 3). D , Mel1 cells (1 × 106 ) were left untreated or treated with filipin, then subjected to a fusion test with R18-exoMel1. A representative fluorescence dequenching curve is shown. Inset , statistical analysis obtained by 30 min kinetic experiments is shown. Values are the means ± S.D. a = p < 0.01 versus control ( n = 3). E , R18-exoMel1ac and R18-exoMel1 (10 μg) were mixed with parental cells at the corresponding pH, and fusion was monitored. A representative fluorescence dequenching curve is shown. Statistical analysis on 5-, 20-, and 30-min kinetic experiments is represented. Values are the means ± S.D. a , p < 0.05 ( n = 3). F , streptavidin blotting is shown. 20 μg of biotinylated exosomes were incubated with parental cells (1 × 106 ) at the corresponding pH ( lane 1 , exoMel1 on Mel1; lane 2 , exoMel1ac on Mel1ac ) or with filipin-treated cells ( lane 3 , exoMel1 on Mel1; lane 4 , exoMel1ac on Mel1ac ) for 1 h at 37 °C. Immunoblotting of nucleolin protein expression represents a control for protein equal loading. A representative Western blot of three independent experiments is shown. Numbers expressed in arbitrary units ( a.u .) represent streptavidin densitometry analysis.

Techniques Used: Confocal Laser Scanning Microscopy, Labeling, Incubation, Fluorescence, Activity Assay, Expressing, Western Blot

12) Product Images from "Elevated cellular cholesterol in Familial Alzheimer’s presenilin 1 mutation is associated with lipid raft localization of β-amyloid precursor protein"

Article Title: Elevated cellular cholesterol in Familial Alzheimer’s presenilin 1 mutation is associated with lipid raft localization of β-amyloid precursor protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0210535

Cholesterol level was higher in the CHO PS1 ΔE9 cells than in the PS1 WT cells. ( a ) Free cholesterol was visualized by filipin staining from the CHO PS1 WT and ΔE9 cells. ( b ) Fluorescence intensities were compared between the PS1 WT (n = 11) and ΔE9 cells (n = 13). ( c ) Total cholesterol levels were measured from cell membrane fractions using the Amplex Red cholesterol assay kit from the PS1 WT and ΔE9 cells (n = 7). Statistical analysis was carried out using one way ANOVA: ***p
Figure Legend Snippet: Cholesterol level was higher in the CHO PS1 ΔE9 cells than in the PS1 WT cells. ( a ) Free cholesterol was visualized by filipin staining from the CHO PS1 WT and ΔE9 cells. ( b ) Fluorescence intensities were compared between the PS1 WT (n = 11) and ΔE9 cells (n = 13). ( c ) Total cholesterol levels were measured from cell membrane fractions using the Amplex Red cholesterol assay kit from the PS1 WT and ΔE9 cells (n = 7). Statistical analysis was carried out using one way ANOVA: ***p

Techniques Used: Staining, Fluorescence, Amplex Red Cholesterol Assay

Elevated cholesterol level in CHO PS1 ΔE9 cells was decreased by the inhibition of cholesterol biosynthesis. ( a ) CHO PS1 WT and ΔE9 cells were pre-treated with 1 μM tebuconazole for 48 h, and free cholesterol was visualized by filipin staining. ( b ) Filipin intensities were compared in the absence (control) and presence of tebuconazole (tebu). PS1 WT control (n = 14), PS1 WT with 10 μM tebuconazole (n = 11), ΔE9 control (n = 10), ΔE9 with 10 μM tebuconazole (n = 10). ( c ) CHO PS1 WT and ΔE9 cells were pre-treated with 0, 1, 5, or 10 μM tebuconazole for 48 h, and total cholesterol level was measured from cell membrane fractions using Amplex Red cholesterol assay kit (n = 8). One way ANOVA: **p
Figure Legend Snippet: Elevated cholesterol level in CHO PS1 ΔE9 cells was decreased by the inhibition of cholesterol biosynthesis. ( a ) CHO PS1 WT and ΔE9 cells were pre-treated with 1 μM tebuconazole for 48 h, and free cholesterol was visualized by filipin staining. ( b ) Filipin intensities were compared in the absence (control) and presence of tebuconazole (tebu). PS1 WT control (n = 14), PS1 WT with 10 μM tebuconazole (n = 11), ΔE9 control (n = 10), ΔE9 with 10 μM tebuconazole (n = 10). ( c ) CHO PS1 WT and ΔE9 cells were pre-treated with 0, 1, 5, or 10 μM tebuconazole for 48 h, and total cholesterol level was measured from cell membrane fractions using Amplex Red cholesterol assay kit (n = 8). One way ANOVA: **p

Techniques Used: Inhibition, Staining, Amplex Red Cholesterol Assay

13) Product Images from "Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus"

Article Title: Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus

Journal: mBio

doi: 10.1128/mBio.01345-18

IFN-β induces cholesterol accumulation in acidic endosomes in A549 cells. (A) A549-WT cells or (B) A549-IFITM3KO cells were left untreated or exposed to IFN-β (10 ng/ml, 16 h) and loaded with LysoTracker Red DND-99 to label acidic endosomes. Unesterified cellular cholesterol was stained using filipin (blue). Filipin pixel intensities were pseudocolored according to the LUT and are shown as a heat map, with the gradient of colors ranging from blue (lowest intensity) to white (highest intensity). Bar, 20 µm. Colocalization coefficients of LysoTracker with filipin were quantitated from z-stacks. Mean values ± SEM were calculated from 35 individual cells per condition from at least two independent experiments. **, P ≤ 0.01; ****, P ≤ 0.0001 (unpaired Student’s t test). (C) A549-WT and A549-IFITM3KO cells were exposed to IFN-β (10 ng/ml, 16 h). The fractions of early endosomes (EE) and late endosomes/lysosomes (LE) were enriched from postnuclear supernatants (PNS) through sucrose gradient separation and analyzed for their cholesterol levels. Data are expressed as mean cholesterol concentrations (in micrograms per milliliter) ± SEM from at least three independent gradients. **, P ≤ 0.01; ns, not significant (two-way ANOVA with Tukey’s multiple-comparison test).
Figure Legend Snippet: IFN-β induces cholesterol accumulation in acidic endosomes in A549 cells. (A) A549-WT cells or (B) A549-IFITM3KO cells were left untreated or exposed to IFN-β (10 ng/ml, 16 h) and loaded with LysoTracker Red DND-99 to label acidic endosomes. Unesterified cellular cholesterol was stained using filipin (blue). Filipin pixel intensities were pseudocolored according to the LUT and are shown as a heat map, with the gradient of colors ranging from blue (lowest intensity) to white (highest intensity). Bar, 20 µm. Colocalization coefficients of LysoTracker with filipin were quantitated from z-stacks. Mean values ± SEM were calculated from 35 individual cells per condition from at least two independent experiments. **, P ≤ 0.01; ****, P ≤ 0.0001 (unpaired Student’s t test). (C) A549-WT and A549-IFITM3KO cells were exposed to IFN-β (10 ng/ml, 16 h). The fractions of early endosomes (EE) and late endosomes/lysosomes (LE) were enriched from postnuclear supernatants (PNS) through sucrose gradient separation and analyzed for their cholesterol levels. Data are expressed as mean cholesterol concentrations (in micrograms per milliliter) ± SEM from at least three independent gradients. **, P ≤ 0.01; ns, not significant (two-way ANOVA with Tukey’s multiple-comparison test).

Techniques Used: Staining

14) Product Images from "Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy"

Article Title: Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy

Journal: Scientific Reports

doi: 10.1038/srep18017

Effect of U18666A and 25-HC in the content and distribution of lysosomal sphingomyelin. ( a ) ASMase +/+ and ASMase −/− PMH were treated overnight with U18666A (1 μg/ml), 25-HC (1 μg/ml) or both and lysosomal colocalization (Lamp2 staining) with cholesterol (filipin staining) and sphingomyelin (lysenin staining) was analysed by inmunofluorescence. ( b,c ) Total sphingomyelin levels in PMH following the different treatments were analysed with a colorimetric detection kit. Scale bar represents 20 μm.
Figure Legend Snippet: Effect of U18666A and 25-HC in the content and distribution of lysosomal sphingomyelin. ( a ) ASMase +/+ and ASMase −/− PMH were treated overnight with U18666A (1 μg/ml), 25-HC (1 μg/ml) or both and lysosomal colocalization (Lamp2 staining) with cholesterol (filipin staining) and sphingomyelin (lysenin staining) was analysed by inmunofluorescence. ( b,c ) Total sphingomyelin levels in PMH following the different treatments were analysed with a colorimetric detection kit. Scale bar represents 20 μm.

Techniques Used: Staining

Human hepatocytes pretreated with amitriptyline exhibit increased LC accumulation, impaired mitophagy and sensitization to APAP-induced cell death. ( a ) Primary human hepatocytes expressing Lamp-GFP and mtKeima were overnight treated with amitriptyline (10 μM) and free cholesterol was stained with filipin. ( b ) 5 images per treatment were analyzed with Image J to assess the percentage of lysosomal colocalization with filipin. Images are representative of 3 independent experiments. Scale bar represents 20 μM. ( c,d ) Lysosomal colocalization with mitochondria of primary human hepatocytes expressing Lamp-GFP and mtKeima pretreated with Amitryptiline (10 μM) with or without 25-HC (1 μg/ml) for 12 hours following exposure to APAP (5 mM) for 3 hours. 5 images per treatment of 3 different experiments were analyzed with Image J to assess the percentage of lysosomal colocalization with mitochondria. ( e ) Cell viability was analysed in human hepatocytes pre-treated with amitryptiline with or withoug 25-HC (1 μg/ml) and exposed APAP 15 mM) for 6 hours. Data are expressed as mean ± SEM of 3 independent experiments. *p
Figure Legend Snippet: Human hepatocytes pretreated with amitriptyline exhibit increased LC accumulation, impaired mitophagy and sensitization to APAP-induced cell death. ( a ) Primary human hepatocytes expressing Lamp-GFP and mtKeima were overnight treated with amitriptyline (10 μM) and free cholesterol was stained with filipin. ( b ) 5 images per treatment were analyzed with Image J to assess the percentage of lysosomal colocalization with filipin. Images are representative of 3 independent experiments. Scale bar represents 20 μM. ( c,d ) Lysosomal colocalization with mitochondria of primary human hepatocytes expressing Lamp-GFP and mtKeima pretreated with Amitryptiline (10 μM) with or without 25-HC (1 μg/ml) for 12 hours following exposure to APAP (5 mM) for 3 hours. 5 images per treatment of 3 different experiments were analyzed with Image J to assess the percentage of lysosomal colocalization with mitochondria. ( e ) Cell viability was analysed in human hepatocytes pre-treated with amitryptiline with or withoug 25-HC (1 μg/ml) and exposed APAP 15 mM) for 6 hours. Data are expressed as mean ± SEM of 3 independent experiments. *p

Techniques Used: Liquid Chromatography, Expressing, Staining

15) Product Images from "Association of tamoxifen resistance and lipid reprogramming in breast cancer"

Article Title: Association of tamoxifen resistance and lipid reprogramming in breast cancer

Journal: BMC Cancer

doi: 10.1186/s12885-018-4757-z

Genes of cholesterol pathway and related lipids are upregulated in the tamoxifen-resistant T-47D cell lines. a Hierarchical clustering and heat map visualization of each parental/resistant cell line pair and patient primary/metastatic tumor. Orange (negative log2-ratio) represents increase and blue (positive log2-ratio) decrease in expression in the resistant cell lines/metastatic tumor. Log2 ratios of > = |2| are displayed in the same color. b Filipin staining reveals an increase in intracellular amounts of free cholesterol in tamoxifen-resistant T-47D cells +/− 1 μM 4-OH- tamoxifen. c Quantification of lipid content in cells grown +/− 1 μM 4-OH-tamoxifen reveals an increase in cholesterol esters and triglycerides in tamoxifen-resistant cells (depicted as colored bars). Only significant differences ( p -value
Figure Legend Snippet: Genes of cholesterol pathway and related lipids are upregulated in the tamoxifen-resistant T-47D cell lines. a Hierarchical clustering and heat map visualization of each parental/resistant cell line pair and patient primary/metastatic tumor. Orange (negative log2-ratio) represents increase and blue (positive log2-ratio) decrease in expression in the resistant cell lines/metastatic tumor. Log2 ratios of > = |2| are displayed in the same color. b Filipin staining reveals an increase in intracellular amounts of free cholesterol in tamoxifen-resistant T-47D cells +/− 1 μM 4-OH- tamoxifen. c Quantification of lipid content in cells grown +/− 1 μM 4-OH-tamoxifen reveals an increase in cholesterol esters and triglycerides in tamoxifen-resistant cells (depicted as colored bars). Only significant differences ( p -value

Techniques Used: Expressing, Staining

16) Product Images from "Coordinate regulation of mutant NPC1 degradation by selective ER autophagy and MARCH6-dependent ERAD"

Article Title: Coordinate regulation of mutant NPC1 degradation by selective ER autophagy and MARCH6-dependent ERAD

Journal: Nature Communications

doi: 10.1038/s41467-018-06115-2

I1061T is degraded by ER-phagy. a CTRL, I1061T, or P1007A/T1036M NPC1 fibroblasts were treated with the following siRNAs: non-targeting (NT), beclin-1 (Bec1), p97, and FAM134B (FAM) at t = 0 and t = 24 h. Lysates were collected at t = 48 h and NPC1 levels analyzed and quantified below. b I1061T NPC1 fibroblasts were treated for 2 sequential days with non-targeting siRNA (siNT) or siRNA FAM134B (siFAM134B), then treated with cycloheximide and serum starved. NPC1 protein levels were analyzed by western blot, quantified at right. c Lysates from a were digested with endoglycosidase H (E), PNGaseF (P), or not treated (NT) and then analyzed by western blot. d I1061T NPC1 fibroblasts were treated with siRNAs as in a . At t = 48 h, cells were stained with filipin. Intensity quantified at right. Scale bar = 50 μm. Data are mean ± s.e.m. from a 3–6; b , c 3 independent experiments. n.s., not significant, * P ≤ 0.05. ** P ≤ 0.01 by a , b ANOVA with a Tukey or b Bonferroni posthoc test; c Student’s t -test. * P ≤ 0.05, a (I10) F = 10.37; b F = 7.45
Figure Legend Snippet: I1061T is degraded by ER-phagy. a CTRL, I1061T, or P1007A/T1036M NPC1 fibroblasts were treated with the following siRNAs: non-targeting (NT), beclin-1 (Bec1), p97, and FAM134B (FAM) at t = 0 and t = 24 h. Lysates were collected at t = 48 h and NPC1 levels analyzed and quantified below. b I1061T NPC1 fibroblasts were treated for 2 sequential days with non-targeting siRNA (siNT) or siRNA FAM134B (siFAM134B), then treated with cycloheximide and serum starved. NPC1 protein levels were analyzed by western blot, quantified at right. c Lysates from a were digested with endoglycosidase H (E), PNGaseF (P), or not treated (NT) and then analyzed by western blot. d I1061T NPC1 fibroblasts were treated with siRNAs as in a . At t = 48 h, cells were stained with filipin. Intensity quantified at right. Scale bar = 50 μm. Data are mean ± s.e.m. from a 3–6; b , c 3 independent experiments. n.s., not significant, * P ≤ 0.05. ** P ≤ 0.01 by a , b ANOVA with a Tukey or b Bonferroni posthoc test; c Student’s t -test. * P ≤ 0.05, a (I10) F = 10.37; b F = 7.45

Techniques Used: Western Blot, Staining

Accumulating I1061T NPC1 does not traffic to lysosomes. a Primary human fibroblasts expressing I1061T or P1007A/T1036M NPC1 were treated for 24 h with vehicle, 10 μM MG132 (MG), 100 nM bafilomycin A1 (Baf), or 1 mM cyclodextrin (Cyclo). Unesterified cholesterol was labeled with filipin (blue), and staining intensity calculated from three independent experiments (16 fields/experiment). Scale bar = 50 μm. Dashed lines outline plasma membrane. Quantified at the bottom. b CTRL, I1061T, or P1007A/T1036M NPC1 fibroblasts were treated for 24 h with vehicle (Veh), 100 nM bafilomycin A1 (Baf), or 100 nM epoxomicin (Epox). Lysates were digested with endoglycosidase H (E), PNGaseF (P), or not treated (NT) and then analyzed by western blot. Data are mean ± s.e.m. n.s., not significant, * P ≤ 0.05, **** P ≤ 0.0001 by ANOVA with Tukey’s posthoc test F = 50.85
Figure Legend Snippet: Accumulating I1061T NPC1 does not traffic to lysosomes. a Primary human fibroblasts expressing I1061T or P1007A/T1036M NPC1 were treated for 24 h with vehicle, 10 μM MG132 (MG), 100 nM bafilomycin A1 (Baf), or 1 mM cyclodextrin (Cyclo). Unesterified cholesterol was labeled with filipin (blue), and staining intensity calculated from three independent experiments (16 fields/experiment). Scale bar = 50 μm. Dashed lines outline plasma membrane. Quantified at the bottom. b CTRL, I1061T, or P1007A/T1036M NPC1 fibroblasts were treated for 24 h with vehicle (Veh), 100 nM bafilomycin A1 (Baf), or 100 nM epoxomicin (Epox). Lysates were digested with endoglycosidase H (E), PNGaseF (P), or not treated (NT) and then analyzed by western blot. Data are mean ± s.e.m. n.s., not significant, * P ≤ 0.05, **** P ≤ 0.0001 by ANOVA with Tukey’s posthoc test F = 50.85

Techniques Used: Expressing, Labeling, Staining, Western Blot

17) Product Images from "Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry"

Article Title: Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry

Journal: Cell Discovery

doi: 10.1038/s41421-018-0034-1

Emetine inhibits EBOV infection in vitro and in vivo. a Dose–response curve showing the inhibition effect of emetine treatment on Ebola VLP entry in HeLa cells. b Dose–response curve showing the inhibition effect of emetine treatment on infection of Ebola live virus in Vero E6 cells. c The survival curve of MA-EBOV infected mouse treated with 1 mg/kg emetine every day. Six to eight week-old female BALB/c mice were randomly assigned into groups ( N = 6 animals). All the mice were challenged with a lethal dose of 1000 times the LD 50 mouse adapted EBOV via IP treatments with either emetine (1 mg/kg/day) or PBS (same volume for the control group) were initiated at 3 h before the challenge and continued for up to 6 days post infection. Survival was monitored for 28 days post infection. d Top, dose–response curve showing the effect of emetine on cholesterol accumulation measured by filipin dye fluorescence intensity (IC 50 = 9.03 µM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with filipin (unesterified free cholesterol, green) and nuclear green (nuclei, blue). e Top, dose–response curve showing the effect of emetine on lipid accumulation measured by Nile red fluorescence intensity (IC 50 = 551 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with Nile red (lipids, yellow) and Hoechst 33342 (nuclei, blue). f Top, dose–response curve showing the effect of emetine on acidic organelle accumulation measured by LysoTracker dye fluorescence intensity (IC 50 = 26.2 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with LysoTracker dye (acidic organelles, red) and Hoechst 33342 (nuclei, blue). All curves represent best fits for calculating the IC 50 values (graph inset). All values represent mean ± SD ( N = 3 replicates). Scale bar=50 µm
Figure Legend Snippet: Emetine inhibits EBOV infection in vitro and in vivo. a Dose–response curve showing the inhibition effect of emetine treatment on Ebola VLP entry in HeLa cells. b Dose–response curve showing the inhibition effect of emetine treatment on infection of Ebola live virus in Vero E6 cells. c The survival curve of MA-EBOV infected mouse treated with 1 mg/kg emetine every day. Six to eight week-old female BALB/c mice were randomly assigned into groups ( N = 6 animals). All the mice were challenged with a lethal dose of 1000 times the LD 50 mouse adapted EBOV via IP treatments with either emetine (1 mg/kg/day) or PBS (same volume for the control group) were initiated at 3 h before the challenge and continued for up to 6 days post infection. Survival was monitored for 28 days post infection. d Top, dose–response curve showing the effect of emetine on cholesterol accumulation measured by filipin dye fluorescence intensity (IC 50 = 9.03 µM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with filipin (unesterified free cholesterol, green) and nuclear green (nuclei, blue). e Top, dose–response curve showing the effect of emetine on lipid accumulation measured by Nile red fluorescence intensity (IC 50 = 551 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with Nile red (lipids, yellow) and Hoechst 33342 (nuclei, blue). f Top, dose–response curve showing the effect of emetine on acidic organelle accumulation measured by LysoTracker dye fluorescence intensity (IC 50 = 26.2 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with LysoTracker dye (acidic organelles, red) and Hoechst 33342 (nuclei, blue). All curves represent best fits for calculating the IC 50 values (graph inset). All values represent mean ± SD ( N = 3 replicates). Scale bar=50 µm

Techniques Used: Infection, In Vitro, In Vivo, Inhibition, Mouse Assay, Fluorescence, Staining

18) Product Images from "STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites"

Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

Journal: The EMBO Journal

doi: 10.15252/embj.201695917

STARD3 needs a functional START domain and ER–endosome contacts to induce cholesterol accumulation in endosomes Schematic representation of the different STARD3 mutants used in the study. The MENTAL domain in light blue contains 4 transmembrane helices (dark blue) and a FFAT motif (red); the START domain in pink contains two essential residues involved in cholesterol binding (M307 and N311). Positions of the epitopes recognized by the rabbit polyclonal pAbMLN64‐Nt‐1611 and pAbMLN64‐Ct‐605 antibodies are shown. Point mutation positions are labeled in black. Western blot analysis of STARD3 expression in the different cell lines. The expression of VAP proteins is unchanged; actin was used as a loading control. *: unspecific band. To mark cholesterol accumulation in endosomes, HeLa/Ctrl (a–c), HeLa/STARD3 (d–f), HeLa/STARD3 ΔSTART (g–i), HeLa/STARD3 MR/ND (j–l), and HeLa/STARD3 FA/YA (m–o) were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe GFP‐D4 (C: green) or filipin (D: Cyan Hot). Nuclei are stained in blue. Higher magnification (2.5×) images of the area outlined in white (a, d, g, j, m) are shown on the right. The GFP‐D4 and STARD3 merged image (C) and the filipin and STARD3 merged image (D) are labeled Overlay. Data information: Scale bars: 10 μm.
Figure Legend Snippet: STARD3 needs a functional START domain and ER–endosome contacts to induce cholesterol accumulation in endosomes Schematic representation of the different STARD3 mutants used in the study. The MENTAL domain in light blue contains 4 transmembrane helices (dark blue) and a FFAT motif (red); the START domain in pink contains two essential residues involved in cholesterol binding (M307 and N311). Positions of the epitopes recognized by the rabbit polyclonal pAbMLN64‐Nt‐1611 and pAbMLN64‐Ct‐605 antibodies are shown. Point mutation positions are labeled in black. Western blot analysis of STARD3 expression in the different cell lines. The expression of VAP proteins is unchanged; actin was used as a loading control. *: unspecific band. To mark cholesterol accumulation in endosomes, HeLa/Ctrl (a–c), HeLa/STARD3 (d–f), HeLa/STARD3 ΔSTART (g–i), HeLa/STARD3 MR/ND (j–l), and HeLa/STARD3 FA/YA (m–o) were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe GFP‐D4 (C: green) or filipin (D: Cyan Hot). Nuclei are stained in blue. Higher magnification (2.5×) images of the area outlined in white (a, d, g, j, m) are shown on the right. The GFP‐D4 and STARD3 merged image (C) and the filipin and STARD3 merged image (D) are labeled Overlay. Data information: Scale bars: 10 μm.

Techniques Used: Functional Assay, Binding Assay, Mutagenesis, Labeling, Western Blot, Expressing, Staining

VAP protein knockdown abolishes STARD3‐mediated cholesterol accumulation in endosomes Western blot analysis of VAP‐A and VAP‐B expression in untreated (NT) HeLa/STARD3 cells or HeLa/STARD3 expressing a control shRNA (shCtrl) or two pairs of shRNAs targeting VAP‐A and VAP‐B (shVAP‐A/B‐α or shVAP‐A/B‐β). Actin was used as a loading control. HeLa cells (a–c), and HeLa/STARD3 cells expressing a control shRNA (shCtrl; d–f) or two pairs of shRNAs targeting VAP‐A and VAP‐B [shVAP‐A/B‐α (g–i) or shVAP‐A/B‐β (j–l)] were labeled with anti‐STARD3 antibodies (magenta) and with the fluorescent cholesterol probe filipin (Cyan Hot). Merged images of filipin and STARD3 signals are shown in (c, f, i and l). Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin signal in HeLa, HeLa/STARD3/shCtrl, HeLa/STARD3/shVAP‐A/B‐α, and HeLa/STARD3/shVAP‐A/B‐β. n = 3 independent experiments. Total number of cells analyzed: HeLa: 84; HeLa/STARD3/shCtrl: 130; HeLa/STARD3/shVAP‐A/B‐α: 109; HeLa/STARD3/shVAP‐A/B‐β: 92. Number of cells analyzed per sample per experiment ≥ 25. Mean ± SD; *** P
Figure Legend Snippet: VAP protein knockdown abolishes STARD3‐mediated cholesterol accumulation in endosomes Western blot analysis of VAP‐A and VAP‐B expression in untreated (NT) HeLa/STARD3 cells or HeLa/STARD3 expressing a control shRNA (shCtrl) or two pairs of shRNAs targeting VAP‐A and VAP‐B (shVAP‐A/B‐α or shVAP‐A/B‐β). Actin was used as a loading control. HeLa cells (a–c), and HeLa/STARD3 cells expressing a control shRNA (shCtrl; d–f) or two pairs of shRNAs targeting VAP‐A and VAP‐B [shVAP‐A/B‐α (g–i) or shVAP‐A/B‐β (j–l)] were labeled with anti‐STARD3 antibodies (magenta) and with the fluorescent cholesterol probe filipin (Cyan Hot). Merged images of filipin and STARD3 signals are shown in (c, f, i and l). Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin signal in HeLa, HeLa/STARD3/shCtrl, HeLa/STARD3/shVAP‐A/B‐α, and HeLa/STARD3/shVAP‐A/B‐β. n = 3 independent experiments. Total number of cells analyzed: HeLa: 84; HeLa/STARD3/shCtrl: 130; HeLa/STARD3/shVAP‐A/B‐α: 109; HeLa/STARD3/shVAP‐A/B‐β: 92. Number of cells analyzed per sample per experiment ≥ 25. Mean ± SD; *** P

Techniques Used: Western Blot, Expressing, shRNA, Labeling, Fluorescence

Cholesterol staining with GFP‐D4 or filipin Plasma membrane cholesterol staining with the GFP‐D4 probe. Live HeLa/Ctrl cells were left untreated (a) or treated with MβCD (b) to remove plasma membrane cholesterol (10 mM in serum‐free medium; 30 min at 37°C), and incubated with GFP‐D4 prior to fixation and nucleus staining (blue). GFP‐D4 highly stained the plasma membrane of untreated cells (a), while almost no staining was present on MβCD‐treated cells (b). Analysis by flow cytometry of plasma cholesterol membrane staining with the GFP‐D4 probe. HeLa cells were either left untreated and unstained (HeLa/no probe), untreated (HeLa + GFP‐D4 probe) or treated with MβCD (HeLa/MβCD treatment + GFP‐D4 probe), and next stained; cells were then analyzed by flow cytometry. These representative histograms display the number of cells analyzed (normalized to mode) as a function of GFP‐D4 fluorescence (log intensity). Note that HeLa cells are strongly labeled with the GFP‐D4 probe; MβCD treatment prior to labeling strongly decreases GFP‐D4 signal intensity. Intracellular cholesterol staining with GFP‐D4. HeLa/Ctrl cells were left untreated (a) or treated with U18666A (1 μg/ml; 1 h at 37°C) to promote intracellular cholesterol accumulation (b). After fixation, cells were permeabilized by freezing in liquid nitrogen and incubated with GFP‐D4. Untreated cells (a) were labeled on small discrete structures by the GFP‐D4 probe; in U18666A‐treated cells (b), cholesterol‐filled endosomes were strongly labeled by the GFP‐D4 probe. Whole‐cell cholesterol staining with filipin on fixed HeLa/Ctrl (a) and HeLa/STARD3 (b) cells. Filipin stains cholesterol in the plasma membrane and in intracellular compartments. Note that HeLa/STARD3 cells display intracellular puncta of filipin staining. Intracellular cholesterol staining with filipin. Live HeLa/Ctrl (a) and HeLa/STARD3 (b) cells were treated with MβCD (10 mM in serum‐free medium; 30 min at 37°C) prior to fixation and filipin staining. MβCD treatment removed cholesterol from the plasma membrane and allowed a better visualization of intracellular cholesterol pools. Data information: Higher magnification (3×) images of the area outlined in white are shown on the right. Scale bars: 10 μm.
Figure Legend Snippet: Cholesterol staining with GFP‐D4 or filipin Plasma membrane cholesterol staining with the GFP‐D4 probe. Live HeLa/Ctrl cells were left untreated (a) or treated with MβCD (b) to remove plasma membrane cholesterol (10 mM in serum‐free medium; 30 min at 37°C), and incubated with GFP‐D4 prior to fixation and nucleus staining (blue). GFP‐D4 highly stained the plasma membrane of untreated cells (a), while almost no staining was present on MβCD‐treated cells (b). Analysis by flow cytometry of plasma cholesterol membrane staining with the GFP‐D4 probe. HeLa cells were either left untreated and unstained (HeLa/no probe), untreated (HeLa + GFP‐D4 probe) or treated with MβCD (HeLa/MβCD treatment + GFP‐D4 probe), and next stained; cells were then analyzed by flow cytometry. These representative histograms display the number of cells analyzed (normalized to mode) as a function of GFP‐D4 fluorescence (log intensity). Note that HeLa cells are strongly labeled with the GFP‐D4 probe; MβCD treatment prior to labeling strongly decreases GFP‐D4 signal intensity. Intracellular cholesterol staining with GFP‐D4. HeLa/Ctrl cells were left untreated (a) or treated with U18666A (1 μg/ml; 1 h at 37°C) to promote intracellular cholesterol accumulation (b). After fixation, cells were permeabilized by freezing in liquid nitrogen and incubated with GFP‐D4. Untreated cells (a) were labeled on small discrete structures by the GFP‐D4 probe; in U18666A‐treated cells (b), cholesterol‐filled endosomes were strongly labeled by the GFP‐D4 probe. Whole‐cell cholesterol staining with filipin on fixed HeLa/Ctrl (a) and HeLa/STARD3 (b) cells. Filipin stains cholesterol in the plasma membrane and in intracellular compartments. Note that HeLa/STARD3 cells display intracellular puncta of filipin staining. Intracellular cholesterol staining with filipin. Live HeLa/Ctrl (a) and HeLa/STARD3 (b) cells were treated with MβCD (10 mM in serum‐free medium; 30 min at 37°C) prior to fixation and filipin staining. MβCD treatment removed cholesterol from the plasma membrane and allowed a better visualization of intracellular cholesterol pools. Data information: Higher magnification (3×) images of the area outlined in white are shown on the right. Scale bars: 10 μm.

Techniques Used: Staining, Incubation, Flow Cytometry, Cytometry, Fluorescence, Labeling

The ER is the main source of cholesterol accumulated by STARD3 in endosomes HeLa/Ctrl and HeLa/STARD3 cells were incubated in normal medium (A), LPDS‐containing medium (B) or normal medium with 50 μM mevinolin and 100 μM mevalonate (C), for 48 h. Cholesterol accumulation in endosomes was detected by filipin staining (Cyan Hot) in endosomes identified by the presence of Lamp1 (red) and STARD3 (magenta). Nuclei were stained in blue. Merged image of filipin and STARD3 signals is shown in (d and h). The subpanels on the right are higher magnification (3.5×) images of the area outlined in white (a, e). The filipin and STARD3 merged image is labeled Overlay. Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin in HeLa/Ctrl and HeLa/STARD3 cells incubated or not in LPDS‐containing medium (D) or treated or not with mevinolin and mevalonate (E). Mean ± SD; n = 5 (D) and n = 4 (E) independent experiments; ** P
Figure Legend Snippet: The ER is the main source of cholesterol accumulated by STARD3 in endosomes HeLa/Ctrl and HeLa/STARD3 cells were incubated in normal medium (A), LPDS‐containing medium (B) or normal medium with 50 μM mevinolin and 100 μM mevalonate (C), for 48 h. Cholesterol accumulation in endosomes was detected by filipin staining (Cyan Hot) in endosomes identified by the presence of Lamp1 (red) and STARD3 (magenta). Nuclei were stained in blue. Merged image of filipin and STARD3 signals is shown in (d and h). The subpanels on the right are higher magnification (3.5×) images of the area outlined in white (a, e). The filipin and STARD3 merged image is labeled Overlay. Scale bars: 10 μm. Relative fluorescence intensity of intracellular filipin in HeLa/Ctrl and HeLa/STARD3 cells incubated or not in LPDS‐containing medium (D) or treated or not with mevinolin and mevalonate (E). Mean ± SD; n = 5 (D) and n = 4 (E) independent experiments; ** P

Techniques Used: Incubation, Staining, Labeling, Fluorescence

Characterization of cholesterol‐enriched endosomes in HeLa/STARD3 cells HeLa/STARD3 cells were co‐labeled with anti‐STARD3 (magenta), with the fluorescent cholesterol probe filipin (Cyan Hot) and with anti‐EEA1 (a–d, green), anti‐Lamp1 (e–h, green), anti‐CD63 (i–l, green), anti‐Rab7 (m–p, green), or anti‐BMP (q–t, green) antibodies. Nuclei were stained in blue. Merged image of magenta and green signals is shown in (c, g, k, o, and s). The subpanels on the right are higher magnification (2.6×) images of the area outlined in white (c, g, k, o, s). Overlays show STARD3 and the endocytic markers merged images. Scale bars: 10 μm. Pearson correlation coefficients between STARD3 and the endocytic markers EEA1, Lamp1, CD63, Rab7, and BMP are shown; each dot represents one single cell; cells originates from three independent experiments (15 cells). The horizontal lines show the mean ± SD. Pearson correlation coefficients between filipin and the early endosome marker EEA1 (left) and with the late endosome marker Rab7 (right). Each dot represents one single cell (left 15 cells, right: 20 cells); cells originate from three independent experiments. The horizontal lines show the mean ± SD. HeLa/STARD3 cells were co‐stained with two fluorescent cholesterol probes (GFP‐D4 in green and filipin in Cyan Hot) and with anti‐STARD3 (magenta). Two similar cells are shown (bottom and top). Nuclei were stained in blue. (d, h) Pixels where the green and the Cyan Hot channels co‐localize are shown in white. The subpanels on the right are higher magnification (2.6×) images of the area outlined in white (a, e). Scale bars: 10 μm. Mander's correlation coefficients between filipin and GFP‐D4 are shown; each dot represents one single cell acquired from three independent experiments (22 cells). M1 corresponds to the fraction of filipin signal overlapping with GFP‐D4 signal. M2 corresponds to the fraction of GFP‐D4 signal overlapping with filipin signal. GFP‐D4‐labeled structures are strongly labeled with filipin, while filipin‐positive structures can be GFP‐D4 positive or GFP‐D4 negative. This illustrates the different properties of the two cholesterol probes: While filipin binds to all free cholesterol, GFP‐D4 only binds to cholesterol‐rich membranes. The horizontal lines show the mean ± SD. Paired two‐tailed t ‐test; *** P
Figure Legend Snippet: Characterization of cholesterol‐enriched endosomes in HeLa/STARD3 cells HeLa/STARD3 cells were co‐labeled with anti‐STARD3 (magenta), with the fluorescent cholesterol probe filipin (Cyan Hot) and with anti‐EEA1 (a–d, green), anti‐Lamp1 (e–h, green), anti‐CD63 (i–l, green), anti‐Rab7 (m–p, green), or anti‐BMP (q–t, green) antibodies. Nuclei were stained in blue. Merged image of magenta and green signals is shown in (c, g, k, o, and s). The subpanels on the right are higher magnification (2.6×) images of the area outlined in white (c, g, k, o, s). Overlays show STARD3 and the endocytic markers merged images. Scale bars: 10 μm. Pearson correlation coefficients between STARD3 and the endocytic markers EEA1, Lamp1, CD63, Rab7, and BMP are shown; each dot represents one single cell; cells originates from three independent experiments (15 cells). The horizontal lines show the mean ± SD. Pearson correlation coefficients between filipin and the early endosome marker EEA1 (left) and with the late endosome marker Rab7 (right). Each dot represents one single cell (left 15 cells, right: 20 cells); cells originate from three independent experiments. The horizontal lines show the mean ± SD. HeLa/STARD3 cells were co‐stained with two fluorescent cholesterol probes (GFP‐D4 in green and filipin in Cyan Hot) and with anti‐STARD3 (magenta). Two similar cells are shown (bottom and top). Nuclei were stained in blue. (d, h) Pixels where the green and the Cyan Hot channels co‐localize are shown in white. The subpanels on the right are higher magnification (2.6×) images of the area outlined in white (a, e). Scale bars: 10 μm. Mander's correlation coefficients between filipin and GFP‐D4 are shown; each dot represents one single cell acquired from three independent experiments (22 cells). M1 corresponds to the fraction of filipin signal overlapping with GFP‐D4 signal. M2 corresponds to the fraction of GFP‐D4 signal overlapping with filipin signal. GFP‐D4‐labeled structures are strongly labeled with filipin, while filipin‐positive structures can be GFP‐D4 positive or GFP‐D4 negative. This illustrates the different properties of the two cholesterol probes: While filipin binds to all free cholesterol, GFP‐D4 only binds to cholesterol‐rich membranes. The horizontal lines show the mean ± SD. Paired two‐tailed t ‐test; *** P

Techniques Used: Labeling, Staining, Marker, Two Tailed Test

Image analysis procedure for filipin staining intensity quantification Fields containing about a dozen of cells labeled with filipin, anti‐STARD3, and propidium iodide (PI) were randomly acquired by confocal microscopy in three‐channel images. Image analysis was performed with Fiji ( http://fiji.sc/ ). Image segmentation was performed on the PI staining image. An intensity threshold was applied to determine the cell contours. Nuclei positions, manually selected on the PI staining image, were used to divide the image into discrete areas with a watershed algorithm (Find maxima). Combination of the thresholded and the segmented particles images allowed to build a segmentation mask where individual cell contours could be determined. To quantify the filipin staining intensity, an intensity threshold was first applied on raw images. This threshold allowed to focus the analysis on filipin accumulation puncta. After applying the segmentation mask onto the filipin staining thresholded image, filipin staining intensity was measured in individual cells.
Figure Legend Snippet: Image analysis procedure for filipin staining intensity quantification Fields containing about a dozen of cells labeled with filipin, anti‐STARD3, and propidium iodide (PI) were randomly acquired by confocal microscopy in three‐channel images. Image analysis was performed with Fiji ( http://fiji.sc/ ). Image segmentation was performed on the PI staining image. An intensity threshold was applied to determine the cell contours. Nuclei positions, manually selected on the PI staining image, were used to divide the image into discrete areas with a watershed algorithm (Find maxima). Combination of the thresholded and the segmented particles images allowed to build a segmentation mask where individual cell contours could be determined. To quantify the filipin staining intensity, an intensity threshold was first applied on raw images. This threshold allowed to focus the analysis on filipin accumulation puncta. After applying the segmentation mask onto the filipin staining thresholded image, filipin staining intensity was measured in individual cells.

Techniques Used: Staining, Labeling, Confocal Microscopy

STARD3 favors cholesterol accumulation in endosomes To follow cholesterol accumulation in late endosomes, HeLa (a–d), HeLa/Ctrl (e–h), and HeLa/STARD3 (i–l) cells were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe GFP‐D4 (green). Nuclei were stained in blue. Merged image of GFP‐D4 and STARD3 signals is shown in (d, h, and l). The subpanels on the right are higher magnification (2.5×) images of the area outlined in white (a, e, i). Overlay indicates GFP‐D4 and STARD3 merged image. (n) Linescan analyses with relative fluorescence intensities of the green, magenta, and red channels along the arrow in (m) (HeLa/STARD3 cell). Black thick lines indicate the positions of late endosomes (LE). Colocalization between GFP‐D4‐positive vesicles and Lamp1 and STARD3 was quantified in HeLa/STARD3 cells (12 cells). Filipin as a second method to follow cholesterol accumulation in late endosomes. HeLa (a–d), HeLa/Ctrl (e–h) and HeLa/STARD3 (i–l) cells were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are stained in blue. Merged image of filipin and STARD3 signals is shown in (d, h and l). Shown on the right are higher magnification (2.5×) images of the area outlined in white (a, e, i). The filipin and STARD3 merged image is labeled Overlay. (n) Linescan analyses with relative fluorescence intensities of the cyan, magenta, and red channels along the arrow in (m) (HeLa/STARD3 cell). Black thick lines indicate the positions of late endosomes. Colocalization between filipin‐positive vesicles and Lamp1 and STARD3 was quantified in HeLa/STARD3 cells (10 cells). Pearson correlation coefficients between STARD3 and GFP‐D4 (left) or filipin (right) staining are shown. Each dot represents a single cell (GFP‐D4: 16 cells; filipin: 24 cells; from three independent experiments). Relative fluorescence intensity of intracellular filipin signals in HeLa, HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 ΔSTART, HeLa/STARD3 MR/ND, and HeLa/STARD3 FA/YA cells. n : number of independent experiments. HeLa, HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 ΔSTART: n = 6; HeLa/STARD3 MR/ND and HeLa/STARD3 FA/YA: n = 3. Total number of cells analyzed: HeLa: 309; HeLa/Ctrl: 234; HeLa/STARD3: 295; HeLa/STARD3 ΔSTART: 238; HeLa/STARD3 MR/ND: 116 and HeLa/STARD3 FA/YA: 137. Number of cells analyzed per sample per experiment ≥ 32. Data information: Scale bars: 10 μm. Mean ± SD; * P
Figure Legend Snippet: STARD3 favors cholesterol accumulation in endosomes To follow cholesterol accumulation in late endosomes, HeLa (a–d), HeLa/Ctrl (e–h), and HeLa/STARD3 (i–l) cells were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe GFP‐D4 (green). Nuclei were stained in blue. Merged image of GFP‐D4 and STARD3 signals is shown in (d, h, and l). The subpanels on the right are higher magnification (2.5×) images of the area outlined in white (a, e, i). Overlay indicates GFP‐D4 and STARD3 merged image. (n) Linescan analyses with relative fluorescence intensities of the green, magenta, and red channels along the arrow in (m) (HeLa/STARD3 cell). Black thick lines indicate the positions of late endosomes (LE). Colocalization between GFP‐D4‐positive vesicles and Lamp1 and STARD3 was quantified in HeLa/STARD3 cells (12 cells). Filipin as a second method to follow cholesterol accumulation in late endosomes. HeLa (a–d), HeLa/Ctrl (e–h) and HeLa/STARD3 (i–l) cells were labeled with anti‐Lamp1 antibodies (red), anti‐STARD3 antibodies (magenta), and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are stained in blue. Merged image of filipin and STARD3 signals is shown in (d, h and l). Shown on the right are higher magnification (2.5×) images of the area outlined in white (a, e, i). The filipin and STARD3 merged image is labeled Overlay. (n) Linescan analyses with relative fluorescence intensities of the cyan, magenta, and red channels along the arrow in (m) (HeLa/STARD3 cell). Black thick lines indicate the positions of late endosomes. Colocalization between filipin‐positive vesicles and Lamp1 and STARD3 was quantified in HeLa/STARD3 cells (10 cells). Pearson correlation coefficients between STARD3 and GFP‐D4 (left) or filipin (right) staining are shown. Each dot represents a single cell (GFP‐D4: 16 cells; filipin: 24 cells; from three independent experiments). Relative fluorescence intensity of intracellular filipin signals in HeLa, HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 ΔSTART, HeLa/STARD3 MR/ND, and HeLa/STARD3 FA/YA cells. n : number of independent experiments. HeLa, HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 ΔSTART: n = 6; HeLa/STARD3 MR/ND and HeLa/STARD3 FA/YA: n = 3. Total number of cells analyzed: HeLa: 309; HeLa/Ctrl: 234; HeLa/STARD3: 295; HeLa/STARD3 ΔSTART: 238; HeLa/STARD3 MR/ND: 116 and HeLa/STARD3 FA/YA: 137. Number of cells analyzed per sample per experiment ≥ 32. Data information: Scale bars: 10 μm. Mean ± SD; * P

Techniques Used: Labeling, Staining, Fluorescence

19) Product Images from "Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer"

Article Title: Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3063

Cholesterol-rich lipid microdomains are involved in α-TEA + TAM circumvention of TAMR . (a) MCF-7/TAMR and MCF-7/HER-2 cells were treated with α-TEA at 20 μ M for 15 hours. Identification of cholesterol-rich microdomains was determined by staining the cells with fluorescein-tagged cholesterol marker Filipin and images viewed by using a fluorescence microscope. (b) MCF-7/TAMR cells were pretreated with 10 μ M exogenous cholesterol for 2 hours, followed by treatment with 20 μ M α-TEA or 20 μ M α-TEA plus 1 μ M TAM for l day. Western blot analyses were performed to determine treatment effects on prosurvival signaling mediators and PARP cleavage. (a, b) Data are representative of three independent experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; MCF-7/HER-2, clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; TAM, tamoxifen; TAMR, tamoxifen resistant.
Figure Legend Snippet: Cholesterol-rich lipid microdomains are involved in α-TEA + TAM circumvention of TAMR . (a) MCF-7/TAMR and MCF-7/HER-2 cells were treated with α-TEA at 20 μ M for 15 hours. Identification of cholesterol-rich microdomains was determined by staining the cells with fluorescein-tagged cholesterol marker Filipin and images viewed by using a fluorescence microscope. (b) MCF-7/TAMR cells were pretreated with 10 μ M exogenous cholesterol for 2 hours, followed by treatment with 20 μ M α-TEA or 20 μ M α-TEA plus 1 μ M TAM for l day. Western blot analyses were performed to determine treatment effects on prosurvival signaling mediators and PARP cleavage. (a, b) Data are representative of three independent experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; MCF-7/HER-2, clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; TAM, tamoxifen; TAMR, tamoxifen resistant.

Techniques Used: Staining, Marker, Fluorescence, Microscopy, Western Blot

TAMR cells in comparison to TAMS cells constitutively express higher levels of prosurvival mediators and cholesterol-rich lipid microdomains . (a) TAMR cell lines MCF-7/TAMR and MCF-7/HER-2, as well as TAMS parental MCF-7/TAMS and MCF-7/Neo cells, were treated with TAM at 1 μ M or VEH (ethanol) in steroid-depleted media containing 17β-estradiol (10 -9 M ) for 2 days. Molecular profile of prosurvival mediators was determined with Western blot analyses. (pHER-1/tHER-1 and pHER-2/tHER-2 were not detected in the MCF-7/TAMS and MCF-7/Neo cells). (b) The expression of lipid microdomains was determined by staining the cells with fluorescein-tagged DiLC16 lipid-raft marker and viewed with a fluorescence microscope. (c) The expression of cholesterol-rich lipid microdomains was determined by staining cells with fluorescein-labeled filipin, a cholesterol marker, and viewed by using a fluorescence microscope. (a-c) Data are representative of a minimum of three independent experiments. DiIC-16, dialkylindocarbocyanine; HER, human epidermal growth factor; MCF-7/HER-2, Clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; MCF-7/TAMS, TAM-sensitive MCF-7/parental; pHER-1, phosphorylated-HER-1; pHER-2, phosphorylated-HER-2; TAM, tamoxifen; TAMR, tamoxifen resistant; TAMS, tamoxifen sensitive; VEH, vehicle control.
Figure Legend Snippet: TAMR cells in comparison to TAMS cells constitutively express higher levels of prosurvival mediators and cholesterol-rich lipid microdomains . (a) TAMR cell lines MCF-7/TAMR and MCF-7/HER-2, as well as TAMS parental MCF-7/TAMS and MCF-7/Neo cells, were treated with TAM at 1 μ M or VEH (ethanol) in steroid-depleted media containing 17β-estradiol (10 -9 M ) for 2 days. Molecular profile of prosurvival mediators was determined with Western blot analyses. (pHER-1/tHER-1 and pHER-2/tHER-2 were not detected in the MCF-7/TAMS and MCF-7/Neo cells). (b) The expression of lipid microdomains was determined by staining the cells with fluorescein-tagged DiLC16 lipid-raft marker and viewed with a fluorescence microscope. (c) The expression of cholesterol-rich lipid microdomains was determined by staining cells with fluorescein-labeled filipin, a cholesterol marker, and viewed by using a fluorescence microscope. (a-c) Data are representative of a minimum of three independent experiments. DiIC-16, dialkylindocarbocyanine; HER, human epidermal growth factor; MCF-7/HER-2, Clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; MCF-7/TAMS, TAM-sensitive MCF-7/parental; pHER-1, phosphorylated-HER-1; pHER-2, phosphorylated-HER-2; TAM, tamoxifen; TAMR, tamoxifen resistant; TAMS, tamoxifen sensitive; VEH, vehicle control.

Techniques Used: Western Blot, Expressing, Staining, Marker, Fluorescence, Microscopy, Labeling

20) Product Images from "IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion"

Article Title: IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004048

IFITM3-mediated restriction of IAV fusion is not related to cholesterol accumulation in endosomes of MDCK cells. (A) Sub-cellular distributions of cholesterol (filipin staining) and IFITM3 (antibody staining) in MDCK-Vector and MDCK-IFITM3 cells. Images show confocal sections through the middle of cells. (B) Filipin staining of MDCK cells pretreated with 20 µM U18666A for 18 h or mock-treated cells. (C) Dose-dependence of U18666A effect on viral fusion. MDCK-Vector cells were pretreated for 18 h with indicated concentrations of U18666A or DMSO (control). BlaM-Vpr-carrying pseudoviruses (MOI = 1) were allowed to fuse with cells for 90 min at 37°C in the presence of U18666A or DMSO. Data are means and SEM from 2 triplicate experiments. (D–F) pH distributions in IAV-carrying endosomes of MDCK cells measured using AF488- and CypHer5E-labeled viruses. Viruses were pre-bound to cells in the cold and incubated at 37°C for 45 min before acquiring images. Calculated pH values are shown for MDCK cells without (D) and with pretreatment with 20 µM U18666A for 18 h (E), as well as for MDCK-IFITM3 cells (F). Data are from 10 image fields each.
Figure Legend Snippet: IFITM3-mediated restriction of IAV fusion is not related to cholesterol accumulation in endosomes of MDCK cells. (A) Sub-cellular distributions of cholesterol (filipin staining) and IFITM3 (antibody staining) in MDCK-Vector and MDCK-IFITM3 cells. Images show confocal sections through the middle of cells. (B) Filipin staining of MDCK cells pretreated with 20 µM U18666A for 18 h or mock-treated cells. (C) Dose-dependence of U18666A effect on viral fusion. MDCK-Vector cells were pretreated for 18 h with indicated concentrations of U18666A or DMSO (control). BlaM-Vpr-carrying pseudoviruses (MOI = 1) were allowed to fuse with cells for 90 min at 37°C in the presence of U18666A or DMSO. Data are means and SEM from 2 triplicate experiments. (D–F) pH distributions in IAV-carrying endosomes of MDCK cells measured using AF488- and CypHer5E-labeled viruses. Viruses were pre-bound to cells in the cold and incubated at 37°C for 45 min before acquiring images. Calculated pH values are shown for MDCK cells without (D) and with pretreatment with 20 µM U18666A for 18 h (E), as well as for MDCK-IFITM3 cells (F). Data are from 10 image fields each.

Techniques Used: Staining, Plasmid Preparation, Labeling, Incubation

IFITM3 restriction of IAV fusion with A549 cells is not related to cholesterol accumulation in endosomes. (A) Sub-cellular distributions of cholesterol and IFITM3 in A549-Vector and A549-IFITM3 cells. Cholesterol and IFITM3 staining was done using filipin and anti-IFITM3 antibody, respectively. Images show confocal slices through the middle section of cells. (B) Filipin staining of A549 cells transduced with shRNA against NPC1 (upper panel) and of cells pretreated with 10 µM U18666A for 18 h (lower panel). (C) Intracellular filipin and IFITM3 signals are poorly correlated. Individual regions of interests within 91 cells were drawn to exclude plasma membrane fluorescence, followed by background subtraction and summation of fluorescence intensity within each region of interest. (D) Western blotting analysis of NPC1 expression in A549 cells transduced with scrambled shRNA (A549.shScr) or with shRNA specific to NPC1 (A549.shNPC1). Tubulin was used as a loading control. (E) IAVpp, VSVpp and EBOVpp fusion with A549.shScr and A549.shNPC1 cells measured by the BlaM assay. Data are means and SEM from 2 triplicate experiments (IAVpp and VSVpp) and 1 triplicate experiment (EBOVpp). (F) Single IAV lipid mixing activity in A549.shScr and A549.shNPC1 cells. Cells were allowed to bind AF488- and vDiD-labeled IAV in the cold and incubated at 37°C for 1 h. The number of vDiD dequenching events was normalized to the total number of cell-bound particles from two experiments (n > 690 particles) for each cell line. Error bars are standard deviations. (G) Dose-dependence of U18666A effect on viral fusion. A549 cells were pre-incubated for 18 h with indicated concentrations of U18666A or DMSO (control). BlaM-Vpr-carrying pseudoviruses (MOI = 1) were allowed to fuse with cells for 90 min at 37°C in the presence of U18666A or DMSO. Data are means and SEM from 2 triplicate experiments. **, P = 0.005.
Figure Legend Snippet: IFITM3 restriction of IAV fusion with A549 cells is not related to cholesterol accumulation in endosomes. (A) Sub-cellular distributions of cholesterol and IFITM3 in A549-Vector and A549-IFITM3 cells. Cholesterol and IFITM3 staining was done using filipin and anti-IFITM3 antibody, respectively. Images show confocal slices through the middle section of cells. (B) Filipin staining of A549 cells transduced with shRNA against NPC1 (upper panel) and of cells pretreated with 10 µM U18666A for 18 h (lower panel). (C) Intracellular filipin and IFITM3 signals are poorly correlated. Individual regions of interests within 91 cells were drawn to exclude plasma membrane fluorescence, followed by background subtraction and summation of fluorescence intensity within each region of interest. (D) Western blotting analysis of NPC1 expression in A549 cells transduced with scrambled shRNA (A549.shScr) or with shRNA specific to NPC1 (A549.shNPC1). Tubulin was used as a loading control. (E) IAVpp, VSVpp and EBOVpp fusion with A549.shScr and A549.shNPC1 cells measured by the BlaM assay. Data are means and SEM from 2 triplicate experiments (IAVpp and VSVpp) and 1 triplicate experiment (EBOVpp). (F) Single IAV lipid mixing activity in A549.shScr and A549.shNPC1 cells. Cells were allowed to bind AF488- and vDiD-labeled IAV in the cold and incubated at 37°C for 1 h. The number of vDiD dequenching events was normalized to the total number of cell-bound particles from two experiments (n > 690 particles) for each cell line. Error bars are standard deviations. (G) Dose-dependence of U18666A effect on viral fusion. A549 cells were pre-incubated for 18 h with indicated concentrations of U18666A or DMSO (control). BlaM-Vpr-carrying pseudoviruses (MOI = 1) were allowed to fuse with cells for 90 min at 37°C in the presence of U18666A or DMSO. Data are means and SEM from 2 triplicate experiments. **, P = 0.005.

Techniques Used: Plasmid Preparation, Staining, Transduction, shRNA, Fluorescence, Western Blot, Expressing, Activity Assay, Labeling, Incubation

Cholesterol accumulation in endosomes of CHO cells does not inhibit viral fusion. (A) Filipin staining of untreated and U18666A-treated (40 µM) CHO cells and of CHO-NPC1 − cells devoid of NPC1. (B) Filipin staining of CHO-Vector and CHO-IFITM3 cells. Images in panels A and B show confocal sections through the middle of cells. (B) Confocal images of CHO-Vector and CHO-IFITM3 cells stained with filipin. (C) IAVpp (MOI = 2), VSVpp (MOI = 1) or EBOVpp (MOI = 2) were pre-bound to CHO or CHO-NPC1 − cells in the cold, incubated at 37°C for 90 min, and the resulting fusion activity was measured by the BlaM assay. Results are plotted as the relative extents of fusion CHO-NPC1 − cells after normalizing to fusion with CHO cells. Control experiments were carried out in 70 mM NH 4 Cl. Data are means and SEM from 3 triplicate experiments. (D) The frequency of lipid mixing in CHO (n = 576) and CHO-NPC1 − cells (n = 1241). Pre-treatment with 0.2 µM BafA1 for 30 min followed by initiation with imaging buffer containing BafA1and 70 mM NH 4 Cl inhibited the fusion activity: only 4 out of 1532 particles underwent lipid mixing. Error bars are standard deviations from at least 4 experiments. (E) Pretreatment of CHO cells with U18666A (40 µM, 8 h) modestly diminishes IAVpp or VSVpp fusion and abrogates EBOVpp fusion, as measured by the BlaM assay. Data are means and SEM from 2 triplicate experiments. ***, P
Figure Legend Snippet: Cholesterol accumulation in endosomes of CHO cells does not inhibit viral fusion. (A) Filipin staining of untreated and U18666A-treated (40 µM) CHO cells and of CHO-NPC1 − cells devoid of NPC1. (B) Filipin staining of CHO-Vector and CHO-IFITM3 cells. Images in panels A and B show confocal sections through the middle of cells. (B) Confocal images of CHO-Vector and CHO-IFITM3 cells stained with filipin. (C) IAVpp (MOI = 2), VSVpp (MOI = 1) or EBOVpp (MOI = 2) were pre-bound to CHO or CHO-NPC1 − cells in the cold, incubated at 37°C for 90 min, and the resulting fusion activity was measured by the BlaM assay. Results are plotted as the relative extents of fusion CHO-NPC1 − cells after normalizing to fusion with CHO cells. Control experiments were carried out in 70 mM NH 4 Cl. Data are means and SEM from 3 triplicate experiments. (D) The frequency of lipid mixing in CHO (n = 576) and CHO-NPC1 − cells (n = 1241). Pre-treatment with 0.2 µM BafA1 for 30 min followed by initiation with imaging buffer containing BafA1and 70 mM NH 4 Cl inhibited the fusion activity: only 4 out of 1532 particles underwent lipid mixing. Error bars are standard deviations from at least 4 experiments. (E) Pretreatment of CHO cells with U18666A (40 µM, 8 h) modestly diminishes IAVpp or VSVpp fusion and abrogates EBOVpp fusion, as measured by the BlaM assay. Data are means and SEM from 2 triplicate experiments. ***, P

Techniques Used: Staining, Plasmid Preparation, Incubation, Activity Assay, Imaging

Related Articles

Centrifugation:

Article Title: Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation
Article Snippet: After centrifugation of 35 min at 250×g , PBMC layer was carefully collected and washed three times with 10 mL of cold PBS at 180×g for 12 min, to get rid of platelets. .. Cells were washed in 2% FBS, 2 µM EDTA PBS (MACS buffer), and then stained with filipin (0.05 mg/mL Sigma-Aldrich, Cat#F9765) at 37 °C for 1 h, CTXb (Cholera Toxin B subunit FITC conjugate—8 µg/mL, Sigma-Aldrich, Cat#C1655) or FITC- conjugated anti-human HLA-DR (BD, Cat#347363) or CD80 (BD, Cat#560926) 4 °C for 30 min in the dark.

Immunocytochemistry:

Article Title: Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content
Article Snippet: Paragraph title: Immunocytochemistry ... To visualize unesterified cholesterol, cells were stained with filipin (125 μg/ml; Sigma-Aldrich) for 1 h at room temperature.

Mass Spectrometry:

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature.

Article Title: Simvastatin promotes NPC1‐mediated free cholesterol efflux from lysosomes through CYP7A1/ LXRα signalling pathway in ox LDL‐loaded macrophages
Article Snippet: The up‐regulation of NPC1 was associated with increased production of 7‐hydroxycholesterol by Cytochrome P450 7A1 (CYP7A1) and subsequent activation of the liver X receptor α (LXRα) signalling pathway. .. All treatment and analysis reagents and biochemical kits utilized in this study were obtained from commercial sources: Cholesterol quantification kit, simvastatin, progesterone, nile red, filipin and 7β‐Hydroxycholesterol (Sigma‐Aldrich, St. Louis, MO, USA); anti‐NPC1 antibody (EMD Millipore, Billerica, MA, USA); Mouse interleukin (IL)‐1 beta/IL‐1F2 Quantikine ELISA kit (R & D Systems, Minneapolis, MN, USA); GenMute™ siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA); oxLDL (TBARS: 29‐44 nmoles MDA/mg; Alfa Aesar, Ward Hill, MA, USA); rabbit anti‐mouse CD68 antibody (Bioss, Woburn, MA, USA); Alexa fluor 633 goat anti‐rat IgG (Life Technologies, Carlsbad, CA, USA); LXRα siRNA, NPC1 siRNA and LAMP‐1 rat monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); cytochrome P450 7A1 siRNA (Origene, Rockville, MD, USA); solid‐phase extraction (SPE) column (Silicycle, Quebec, QC, Canada); 22(S)‐hydroxycholesterol (D7) (Avanti Polar Lipids, Alabaster, AL, USA); HPLC column, XTerra RP18, 2.1 × 150 mm, 5 μM (Waters Corporation, Milford, MS, USA); and lysosome enrichment kit (Thermo Scientific, Waltham, MA, USA). .. C57BL/6J mice were obtained from the Jackson Laboratory (Bar harbor, ME, USA) and cared for under housing and animal protocols approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.

Cell Isolation:

Article Title: Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation
Article Snippet: Cells were washed in 2% FBS, 2 µM EDTA PBS (MACS buffer), and then stained with filipin (0.05 mg/mL Sigma-Aldrich, Cat#F9765) at 37 °C for 1 h, CTXb (Cholera Toxin B subunit FITC conjugate—8 µg/mL, Sigma-Aldrich, Cat#C1655) or FITC- conjugated anti-human HLA-DR (BD, Cat#347363) or CD80 (BD, Cat#560926) 4 °C for 30 min in the dark. .. Following staining, cells were washed with 2% FBS, 2 µM EDTA PBS (MACS buffer) and analysed by flow cytometry.

Blocking Assay:

Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites
Article Snippet: Cells were then fixed for 10 min at RT with 4% paraformaldehyde in phosphate‐buffered saline (PBS). .. Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies. .. After three washes in PBS, cells were incubated for 1 h with Cy3‐ and Alexa488‐conjugated secondary antibodies (Jackson ImmunoResearch and Invitrogen‐Molecular Probes, respectively).

Enzyme-linked Immunosorbent Assay:

Article Title: Simvastatin promotes NPC1‐mediated free cholesterol efflux from lysosomes through CYP7A1/ LXRα signalling pathway in ox LDL‐loaded macrophages
Article Snippet: The up‐regulation of NPC1 was associated with increased production of 7‐hydroxycholesterol by Cytochrome P450 7A1 (CYP7A1) and subsequent activation of the liver X receptor α (LXRα) signalling pathway. .. All treatment and analysis reagents and biochemical kits utilized in this study were obtained from commercial sources: Cholesterol quantification kit, simvastatin, progesterone, nile red, filipin and 7β‐Hydroxycholesterol (Sigma‐Aldrich, St. Louis, MO, USA); anti‐NPC1 antibody (EMD Millipore, Billerica, MA, USA); Mouse interleukin (IL)‐1 beta/IL‐1F2 Quantikine ELISA kit (R & D Systems, Minneapolis, MN, USA); GenMute™ siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA); oxLDL (TBARS: 29‐44 nmoles MDA/mg; Alfa Aesar, Ward Hill, MA, USA); rabbit anti‐mouse CD68 antibody (Bioss, Woburn, MA, USA); Alexa fluor 633 goat anti‐rat IgG (Life Technologies, Carlsbad, CA, USA); LXRα siRNA, NPC1 siRNA and LAMP‐1 rat monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); cytochrome P450 7A1 siRNA (Origene, Rockville, MD, USA); solid‐phase extraction (SPE) column (Silicycle, Quebec, QC, Canada); 22(S)‐hydroxycholesterol (D7) (Avanti Polar Lipids, Alabaster, AL, USA); HPLC column, XTerra RP18, 2.1 × 150 mm, 5 μM (Waters Corporation, Milford, MS, USA); and lysosome enrichment kit (Thermo Scientific, Waltham, MA, USA). .. C57BL/6J mice were obtained from the Jackson Laboratory (Bar harbor, ME, USA) and cared for under housing and animal protocols approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.

Incubation:

Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites
Article Snippet: Cells were then fixed for 10 min at RT with 4% paraformaldehyde in phosphate‐buffered saline (PBS). .. Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies. .. After three washes in PBS, cells were incubated for 1 h with Cy3‐ and Alexa488‐conjugated secondary antibodies (Jackson ImmunoResearch and Invitrogen‐Molecular Probes, respectively).

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: After fixation, cells were washed with PBS and incubated with 1.5 g glycine in PBS 10 min at RT. .. Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT.

Article Title: cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells
Article Snippet: Cells were rinsed three times with PBS and then fixed with 2% paraformaldehyde for ∼30 min. .. Cells were rinsed once more with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h on a shaker in the dark. .. Filipin was dissolved freshly in dimethylsulfoxide before each experiment.

Article Title: Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives
Article Snippet: The MIC value was determined to be the lowest concentration of antibiotic, which inhibited the growth of the yeast strain and could be determined by eye on the 96-well plate after an incubation of 24 h at 30°C. .. Commercial filipin III (Sigma) was used as control.

Article Title: A Murine Niemann-Pick C1 I1061T Knock-In Model Recapitulates the Pathological Features of the Most Prevalent Human Disease Allele
Article Snippet: Twenty-four hours after plating, cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Tween 20 with 10% normal goat serum in PBS for 30 min. .. Cells were stained with α-NPC1 antibody (rabbit, 1:250, in house), α-LAMP1 (rat, 1:500, University of Iowa Hybridoma Bank), and filipin (0.05 mg/ml, Sigma) for 1 h. Cells were then incubated with rabbit Alexa Fluor 594 and rat Alexa Fluor 488 for 1 h and mounted with Slowfade (Invitrogen). .. Images were captured with the 63× objective on a laser-scanning confocal microscope (LSM 700 URGB two-channel system; Carl Zeiss) with 405, 488, 555, and 635 nm solid-state lasers coupled to an Axio Imager M2 microscope with a motorized stage and Zen software.

Article Title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis
Article Snippet: Then, preparations were incubated at 37°C for 1 h with α-EhNPC1 (1:100) or α-EhNPC2 (1:100) or α-EhADH (1:500) or α-EhRab7A (1:500) or α-Gal/GalNAc lectin (1:50) (kindly provided by Dr. W. Petri, University of Virginia, Charlottesville, USA) [ ] or α-LBPA (1:30) [ , ] or α-EhSERCA (1:1000) antibodies; followed by extensive washing and incubation for 1 h with species-specific FITC-, TRITC- or Cy5- labeled secondary antibodies (Zymed; 1:100) as appropriate. .. In some cases, nuclei were counterstained with propidium iodide (0.1 μg/ml) (Sigma) for 5 min. For cholesterol detection, cells were stained with 250 μg/ml filipin (Sigma).

Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts
Article Snippet: Primary and secondary antibodies were diluted in 3% bovine serum albumin (BSA) in PBS and incubated on the cells for 1 h. Goat anti-rabbit, -rat, or -mouse conjugated to Alexa Fluor 488 or 594 (Molecular Probes, Eugene, OR) were used as secondary antibodies. .. For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS. .. After filipin staining, immunofluorescence labeling was performed without a separate permeabilization step.

Article Title: Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells
Article Snippet: Cells were rinsed three times with PBS (138 mM NaCl, 15 mM Na2 HPO4 , 1.5 mM KCl, and 2.5 mM KH2 PO4 ) and then fixed using 2% paraformaldehyde for ∼30 min. .. Cells were rinsed three more times with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h on a shaker in the dark. .. Filipin is a bacterial product that binds to free cholesterol and fluoresces in the ultraviolet range when bound.

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Cells were regularly passaged to maintain adequate growth and were passaged at least five times before trypsinization and plating on either graphene-coated or bare glass coverslips (25 μL or ~2 × 106 cells per coverslip). .. Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A.

Activity Assay:

Article Title: A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking
Article Snippet: Protein A–Sepharose CL-4B, [1α,2α(n)-3 H]-Cholesterol (41.0 Ci/mmol), and [1-14 C]-Palmitate (specific activity, 51 mCi/mmol) were obtained from GE Healthcare. .. Compactin (mevastatin), mevalonate, filipin, saponin, and protease inhibitor cocktail were obtained from Sigma-Aldrich.

Infection:

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. For labeling of host cell cholesterol pool by fluorescent cholesterol species, macrophages were cultured as indicated above.

Expressing:

Article Title: IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion
Article Snippet: Vectors expressing phCMV-GPc Lassa and pcDNA3.1-Ebola GP (Zaire) were gifts from Dr. F.-L. Cosset (Université de Lyon, France) and Dr. L. Rong (University of Illinois) , respectively. .. Poly-L-lysine, filipin, sulphorhodamine B Bafilomycin A1 and the Cholesterol Kit were from Sigma-Aldrich.

Western Blot:

Article Title: Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism
Article Snippet: Filipin III was from Sigma. .. For the flow cytometric analysis, anti-mCD4 (RM4-5), anti-mCD8 (53-6.7), anti-mCD3ε (145-2C11), anti-IFNγ (XMG1.2), anti-TNFα (MP6-XT22), anti-granzyme B (NGZB), anti-CD44 (IM7), anti-CD69 (H1.2F3), anti-PD-1 (J43), anti-CTLA-4 (UC10-4B9), anti-Ki-67 (16A8), anti-FoxP3 (FJK-16 s), anti-Gr1 (RB6-8C5), anti-CD11b (M1/70) and anti-CD45 (30-F11) were purchased from eBioscience.

High Performance Liquid Chromatography:

Article Title: Simvastatin promotes NPC1‐mediated free cholesterol efflux from lysosomes through CYP7A1/ LXRα signalling pathway in ox LDL‐loaded macrophages
Article Snippet: The up‐regulation of NPC1 was associated with increased production of 7‐hydroxycholesterol by Cytochrome P450 7A1 (CYP7A1) and subsequent activation of the liver X receptor α (LXRα) signalling pathway. .. All treatment and analysis reagents and biochemical kits utilized in this study were obtained from commercial sources: Cholesterol quantification kit, simvastatin, progesterone, nile red, filipin and 7β‐Hydroxycholesterol (Sigma‐Aldrich, St. Louis, MO, USA); anti‐NPC1 antibody (EMD Millipore, Billerica, MA, USA); Mouse interleukin (IL)‐1 beta/IL‐1F2 Quantikine ELISA kit (R & D Systems, Minneapolis, MN, USA); GenMute™ siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA); oxLDL (TBARS: 29‐44 nmoles MDA/mg; Alfa Aesar, Ward Hill, MA, USA); rabbit anti‐mouse CD68 antibody (Bioss, Woburn, MA, USA); Alexa fluor 633 goat anti‐rat IgG (Life Technologies, Carlsbad, CA, USA); LXRα siRNA, NPC1 siRNA and LAMP‐1 rat monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); cytochrome P450 7A1 siRNA (Origene, Rockville, MD, USA); solid‐phase extraction (SPE) column (Silicycle, Quebec, QC, Canada); 22(S)‐hydroxycholesterol (D7) (Avanti Polar Lipids, Alabaster, AL, USA); HPLC column, XTerra RP18, 2.1 × 150 mm, 5 μM (Waters Corporation, Milford, MS, USA); and lysosome enrichment kit (Thermo Scientific, Waltham, MA, USA). .. C57BL/6J mice were obtained from the Jackson Laboratory (Bar harbor, ME, USA) and cared for under housing and animal protocols approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.

Electron Microscopy:

Article Title: Annexin A6-Induced Alterations in Cholesterol Transport and Caveolin Export from the Golgi Complex
Article Snippet: Nutrient Mixture Ham’s F-12, DMEM, RPMI-1640, filipin, Cyhx and water-soluble cholesterol, saponin, U18666A and CD were from Sigma. .. Nutrient Mixture Ham’s F-12, DMEM, RPMI-1640, filipin, Cyhx and water-soluble cholesterol, saponin, U18666A and CD were from Sigma.

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A.

Transfection:

Article Title: Simvastatin promotes NPC1‐mediated free cholesterol efflux from lysosomes through CYP7A1/ LXRα signalling pathway in ox LDL‐loaded macrophages
Article Snippet: The up‐regulation of NPC1 was associated with increased production of 7‐hydroxycholesterol by Cytochrome P450 7A1 (CYP7A1) and subsequent activation of the liver X receptor α (LXRα) signalling pathway. .. All treatment and analysis reagents and biochemical kits utilized in this study were obtained from commercial sources: Cholesterol quantification kit, simvastatin, progesterone, nile red, filipin and 7β‐Hydroxycholesterol (Sigma‐Aldrich, St. Louis, MO, USA); anti‐NPC1 antibody (EMD Millipore, Billerica, MA, USA); Mouse interleukin (IL)‐1 beta/IL‐1F2 Quantikine ELISA kit (R & D Systems, Minneapolis, MN, USA); GenMute™ siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA); oxLDL (TBARS: 29‐44 nmoles MDA/mg; Alfa Aesar, Ward Hill, MA, USA); rabbit anti‐mouse CD68 antibody (Bioss, Woburn, MA, USA); Alexa fluor 633 goat anti‐rat IgG (Life Technologies, Carlsbad, CA, USA); LXRα siRNA, NPC1 siRNA and LAMP‐1 rat monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); cytochrome P450 7A1 siRNA (Origene, Rockville, MD, USA); solid‐phase extraction (SPE) column (Silicycle, Quebec, QC, Canada); 22(S)‐hydroxycholesterol (D7) (Avanti Polar Lipids, Alabaster, AL, USA); HPLC column, XTerra RP18, 2.1 × 150 mm, 5 μM (Waters Corporation, Milford, MS, USA); and lysosome enrichment kit (Thermo Scientific, Waltham, MA, USA). .. C57BL/6J mice were obtained from the Jackson Laboratory (Bar harbor, ME, USA) and cared for under housing and animal protocols approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.

Protease Inhibitor:

Article Title: A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking
Article Snippet: LDL was subfractionated by density gradient ultracentrifugation from the plasma of healthy male volunteers ( ). .. Compactin (mevastatin), mevalonate, filipin, saponin, and protease inhibitor cocktail were obtained from Sigma-Aldrich. .. Yeast extract, peptone, glucose, tryptone, and yeast nitrogen base were obtained from BD.

Cell Culture:

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. After staining, cells were washed three times with PBS and the cover slips were mounted in Vectashield HardSet Mounting Medium (Vector Laboratories, Burlingame, USA).

Article Title: A Murine Niemann-Pick C1 I1061T Knock-In Model Recapitulates the Pathological Features of the Most Prevalent Human Disease Allele
Article Snippet: Paragraph title: Cell culture immunofluorescence. ... Cells were stained with α-NPC1 antibody (rabbit, 1:250, in house), α-LAMP1 (rat, 1:500, University of Iowa Hybridoma Bank), and filipin (0.05 mg/ml, Sigma) for 1 h. Cells were then incubated with rabbit Alexa Fluor 594 and rat Alexa Fluor 488 for 1 h and mounted with Slowfade (Invitrogen).

Article Title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis
Article Snippet: In some cases, nuclei were counterstained with propidium iodide (0.1 μg/ml) (Sigma) for 5 min. For cholesterol detection, cells were stained with 250 μg/ml filipin (Sigma). .. For acidic vacuoles detection, fixed trophozoites were incubated with 2 μg/ml of Lysotracker (Molecular Probes) for 2 h at 37°C.

Hemagglutination Assay:

Article Title: IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion
Article Snippet: The pCAGGS vectors encoding influenza H1N1 WSN HA and NA were provided by Donna Tscerne and Peter Palese, and the pCAGGS BlaM1 (WSN) plasmid was a gift from Dr. A. Garcia-Sastre (Mount Sinai). .. Poly-L-lysine, filipin, sulphorhodamine B Bafilomycin A1 and the Cholesterol Kit were from Sigma-Aldrich.

other:

Article Title: Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism
Article Snippet: DMSO, tamoxifen, filipin, phenylarsine oxide (PAO) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich.

Article Title: Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells
Article Snippet: TNBC cells were treated with the lipid raft disrupting agents MβCD, nystatin and filipin III (Sigma, St. Louis, USA) at concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mM (0.1–0.5 mM) for 1, 24, and 48 hours.

Imaging:

Article Title: Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway
Article Snippet: Cellular cholesterol was visualised using filipin (Sigma). .. Fixed cells were incubated with 1ml filipin working solution (0.05mg/ml in PBS with 0.2% Triton X100) for 1h at room temperature, before being washed with 3×1ml PBS.

Immunofluorescence:

Article Title: A Murine Niemann-Pick C1 I1061T Knock-In Model Recapitulates the Pathological Features of the Most Prevalent Human Disease Allele
Article Snippet: Paragraph title: Cell culture immunofluorescence. ... Cells were stained with α-NPC1 antibody (rabbit, 1:250, in house), α-LAMP1 (rat, 1:500, University of Iowa Hybridoma Bank), and filipin (0.05 mg/ml, Sigma) for 1 h. Cells were then incubated with rabbit Alexa Fluor 594 and rat Alexa Fluor 488 for 1 h and mounted with Slowfade (Invitrogen).

Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts
Article Snippet: Paragraph title: Immunofluorescence ... For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS.

Magnetic Cell Separation:

Article Title: Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation
Article Snippet: After 5 days, LPS (1 µg/mL, Sigma-Aldrich, Cat#L2630) was added and cells collected 48 h later. .. Cells were washed in 2% FBS, 2 µM EDTA PBS (MACS buffer), and then stained with filipin (0.05 mg/mL Sigma-Aldrich, Cat#F9765) at 37 °C for 1 h, CTXb (Cholera Toxin B subunit FITC conjugate—8 µg/mL, Sigma-Aldrich, Cat#C1655) or FITC- conjugated anti-human HLA-DR (BD, Cat#347363) or CD80 (BD, Cat#560926) 4 °C for 30 min in the dark. .. Following staining, cells were washed with 2% FBS, 2 µM EDTA PBS (MACS buffer) and analysed by flow cytometry.

MTT Assay:

Article Title: Imipramine Inhibits Chikungunya Virus Replication in Human Skin Fibroblasts through Interference with Intracellular Cholesterol Trafficking
Article Snippet: Secondary antibodies (HRP conjugated goat anti-mouse or -rabbit) were purchased from Jackson Laboratories (Westgrove, PA). .. Mouse monoclonal anti-β –actin antibody, U18666A (3 β-[2-(diethylamino)ethoxy]an- drost-5-en-17-one), imipramine hydrochloride, filipin and thiazolyl blue tetrazolium bromide (MTT) were from Sigma-Aldrich (St Louis, MO). .. Cell viability was determined using MTT-based assay.

Fluorescence:

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Paragraph title: Fluorescence microscopy ... Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT.

Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts
Article Snippet: For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS. .. Acidic compartments were labeled by incubating cells in Lysotracker Red or DAMP ( N -(3-((2,4-dinitrophenyl)amino) propyl)- N -(3-aminopropyl) methylamine) (Molecular Probes).

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A.

Isolation:

Article Title: Annexin A6-Induced Alterations in Cholesterol Transport and Caveolin Export from the Golgi Complex
Article Snippet: Nutrient Mixture Ham’s F-12, DMEM, RPMI-1640, filipin, Cyhx and water-soluble cholesterol, saponin, U18666A and CD were from Sigma. .. Nutrient Mixture Ham’s F-12, DMEM, RPMI-1640, filipin, Cyhx and water-soluble cholesterol, saponin, U18666A and CD were from Sigma.

Article Title: A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking
Article Snippet: Lipoprotein-deficient serum was isolated from NCS by ultracentrifugation as described previously ( ). .. Compactin (mevastatin), mevalonate, filipin, saponin, and protease inhibitor cocktail were obtained from Sigma-Aldrich.

Article Title: Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation
Article Snippet: Paragraph title: Isolation and culture of MDCs and T cells from human blood ... Cells were washed in 2% FBS, 2 µM EDTA PBS (MACS buffer), and then stained with filipin (0.05 mg/mL Sigma-Aldrich, Cat#F9765) at 37 °C for 1 h, CTXb (Cholera Toxin B subunit FITC conjugate—8 µg/mL, Sigma-Aldrich, Cat#C1655) or FITC- conjugated anti-human HLA-DR (BD, Cat#347363) or CD80 (BD, Cat#560926) 4 °C for 30 min in the dark.

Flow Cytometry:

Article Title: Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism
Article Snippet: Filipin III was from Sigma. .. Filipin III was from Sigma.

Microscopy:

Article Title: Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content
Article Snippet: To visualize unesterified cholesterol, cells were stained with filipin (125 μg/ml; Sigma-Aldrich) for 1 h at room temperature. .. To visualize unesterified cholesterol, cells were stained with filipin (125 μg/ml; Sigma-Aldrich) for 1 h at room temperature.

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Paragraph title: Fluorescence microscopy ... Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT.

Article Title: cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells
Article Snippet: Cells were rinsed once more with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h on a shaker in the dark. .. Cells were rinsed once more with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h on a shaker in the dark.

Article Title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis
Article Snippet: In some cases, nuclei were counterstained with propidium iodide (0.1 μg/ml) (Sigma) for 5 min. For cholesterol detection, cells were stained with 250 μg/ml filipin (Sigma). .. We also performed experiments using 12 h ABS-starved trophozoites (incubated in TYI medium), cultured on coverslips, and challenged with 100 μl of ABS for 0.5 to 7 min at 37°C, then, samples were processed as above.

Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts
Article Snippet: For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS. .. Acidic compartments were labeled by incubating cells in Lysotracker Red or DAMP ( N -(3-((2,4-dinitrophenyl)amino) propyl)- N -(3-aminopropyl) methylamine) (Molecular Probes).

Article Title: Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway
Article Snippet: Cellular cholesterol was visualised using filipin (Sigma). .. Fixed cells were incubated with 1ml filipin working solution (0.05mg/ml in PBS with 0.2% Triton X100) for 1h at room temperature, before being washed with 3×1ml PBS.

Mouse Assay:

Article Title: Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism
Article Snippet: Paragraph title: Reagents and mice ... Filipin III was from Sigma.

Labeling:

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. After staining, cells were washed three times with PBS and the cover slips were mounted in Vectashield HardSet Mounting Medium (Vector Laboratories, Burlingame, USA).

Article Title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis
Article Snippet: Then, preparations were incubated at 37°C for 1 h with α-EhNPC1 (1:100) or α-EhNPC2 (1:100) or α-EhADH (1:500) or α-EhRab7A (1:500) or α-Gal/GalNAc lectin (1:50) (kindly provided by Dr. W. Petri, University of Virginia, Charlottesville, USA) [ ] or α-LBPA (1:30) [ , ] or α-EhSERCA (1:1000) antibodies; followed by extensive washing and incubation for 1 h with species-specific FITC-, TRITC- or Cy5- labeled secondary antibodies (Zymed; 1:100) as appropriate. .. In some cases, nuclei were counterstained with propidium iodide (0.1 μg/ml) (Sigma) for 5 min. For cholesterol detection, cells were stained with 250 μg/ml filipin (Sigma).

Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts
Article Snippet: For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS. .. After filipin staining, immunofluorescence labeling was performed without a separate permeabilization step.

Confocal Microscopy:

Article Title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis
Article Snippet: Paragraph title: Laser confocal microscopy experiments ... In some cases, nuclei were counterstained with propidium iodide (0.1 μg/ml) (Sigma) for 5 min. For cholesterol detection, cells were stained with 250 μg/ml filipin (Sigma).

Chloramphenicol Acetyltransferase Assay:

Article Title: Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation
Article Snippet: After 5 days, LPS (1 µg/mL, Sigma-Aldrich, Cat#L2630) was added and cells collected 48 h later. .. Cells were washed in 2% FBS, 2 µM EDTA PBS (MACS buffer), and then stained with filipin (0.05 mg/mL Sigma-Aldrich, CatF9765) at 37 °C for 1 h, CTXb (Cholera Toxin B subunit FITC conjugate—8 µg/mL, Sigma-Aldrich, Cat#C1655) or FITC- conjugated anti-human HLA-DR (BD, Cat#347363) or CD80 (BD, Cat#560926) 4 °C for 30 min in the dark. .. Following staining, cells were washed with 2% FBS, 2 µM EDTA PBS (MACS buffer) and analysed by flow cytometry.

Plasmid Preparation:

Article Title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis
Article Snippet: In some cases, nuclei were counterstained with propidium iodide (0.1 μg/ml) (Sigma) for 5 min. For cholesterol detection, cells were stained with 250 μg/ml filipin (Sigma). .. We also performed experiments using 12 h ABS-starved trophozoites (incubated in TYI medium), cultured on coverslips, and challenged with 100 μl of ABS for 0.5 to 7 min at 37°C, then, samples were processed as above.

Article Title: IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion
Article Snippet: The pCAGGS vectors encoding influenza H1N1 WSN HA and NA were provided by Donna Tscerne and Peter Palese, and the pCAGGS BlaM1 (WSN) plasmid was a gift from Dr. A. Garcia-Sastre (Mount Sinai). .. Poly-L-lysine, filipin, sulphorhodamine B Bafilomycin A1 and the Cholesterol Kit were from Sigma-Aldrich.

Software:

Article Title: Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content
Article Snippet: To visualize unesterified cholesterol, cells were stained with filipin (125 μg/ml; Sigma-Aldrich) for 1 h at room temperature. .. To visualize unesterified cholesterol, cells were stained with filipin (125 μg/ml; Sigma-Aldrich) for 1 h at room temperature.

Article Title: cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells
Article Snippet: Cells were rinsed once more with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h on a shaker in the dark. .. Cells were rinsed once more with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h on a shaker in the dark.

Article Title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis
Article Snippet: In some cases, nuclei were counterstained with propidium iodide (0.1 μg/ml) (Sigma) for 5 min. For cholesterol detection, cells were stained with 250 μg/ml filipin (Sigma). .. We also performed experiments using 12 h ABS-starved trophozoites (incubated in TYI medium), cultured on coverslips, and challenged with 100 μl of ABS for 0.5 to 7 min at 37°C, then, samples were processed as above.

Article Title: Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway
Article Snippet: Cellular cholesterol was visualised using filipin (Sigma). .. Fixed cells were incubated with 1ml filipin working solution (0.05mg/ml in PBS with 0.2% Triton X100) for 1h at room temperature, before being washed with 3×1ml PBS.

In Vitro:

Article Title: A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking
Article Snippet: Compactin (mevastatin), mevalonate, filipin, saponin, and protease inhibitor cocktail were obtained from Sigma-Aldrich. .. Compactin (mevastatin), mevalonate, filipin, saponin, and protease inhibitor cocktail were obtained from Sigma-Aldrich.

Concentration Assay:

Article Title: Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives
Article Snippet: The MIC value was determined to be the lowest concentration of antibiotic, which inhibited the growth of the yeast strain and could be determined by eye on the 96-well plate after an incubation of 24 h at 30°C. .. Commercial filipin III (Sigma) was used as control.

Staining:

Article Title: STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites
Article Snippet: Paragraph title: Filipin staining and quantification ... Cells were incubated with a solution of filipin (0.1 mg/ml, F‐9765 Sigma) for 30 min. After washing and blocking in 1% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies.

Article Title: Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content
Article Snippet: Antibodies against LAMP-2 (Southern Biotech, Birmingham, AL, USA), followed by antibodies conjugated to Alexa Fluor (Molecular Probes), were used. .. To visualize unesterified cholesterol, cells were stained with filipin (125 μg/ml; Sigma-Aldrich) for 1 h at room temperature. .. Cover slips were washed and mounted using Prolong gold (Invitrogen).

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: After fixation, cells were washed with PBS and incubated with 1.5 g glycine in PBS 10 min at RT. .. Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. After staining, cells were washed three times with PBS and the cover slips were mounted in Vectashield HardSet Mounting Medium (Vector Laboratories, Burlingame, USA).

Article Title: cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells
Article Snippet: Paragraph title: Filipin staining. ... Cells were rinsed once more with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h on a shaker in the dark.

Article Title: A Murine Niemann-Pick C1 I1061T Knock-In Model Recapitulates the Pathological Features of the Most Prevalent Human Disease Allele
Article Snippet: Twenty-four hours after plating, cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Tween 20 with 10% normal goat serum in PBS for 30 min. .. Cells were stained with α-NPC1 antibody (rabbit, 1:250, in house), α-LAMP1 (rat, 1:500, University of Iowa Hybridoma Bank), and filipin (0.05 mg/ml, Sigma) for 1 h. Cells were then incubated with rabbit Alexa Fluor 594 and rat Alexa Fluor 488 for 1 h and mounted with Slowfade (Invitrogen). .. Images were captured with the 63× objective on a laser-scanning confocal microscope (LSM 700 URGB two-channel system; Carl Zeiss) with 405, 488, 555, and 635 nm solid-state lasers coupled to an Axio Imager M2 microscope with a motorized stage and Zen software.

Article Title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis
Article Snippet: Then, preparations were incubated at 37°C for 1 h with α-EhNPC1 (1:100) or α-EhNPC2 (1:100) or α-EhADH (1:500) or α-EhRab7A (1:500) or α-Gal/GalNAc lectin (1:50) (kindly provided by Dr. W. Petri, University of Virginia, Charlottesville, USA) [ ] or α-LBPA (1:30) [ , ] or α-EhSERCA (1:1000) antibodies; followed by extensive washing and incubation for 1 h with species-specific FITC-, TRITC- or Cy5- labeled secondary antibodies (Zymed; 1:100) as appropriate. .. In some cases, nuclei were counterstained with propidium iodide (0.1 μg/ml) (Sigma) for 5 min. For cholesterol detection, cells were stained with 250 μg/ml filipin (Sigma). .. For acidic vacuoles detection, fixed trophozoites were incubated with 2 μg/ml of Lysotracker (Molecular Probes) for 2 h at 37°C.

Article Title: Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts
Article Snippet: Primary and secondary antibodies were diluted in 3% bovine serum albumin (BSA) in PBS and incubated on the cells for 1 h. Goat anti-rabbit, -rat, or -mouse conjugated to Alexa Fluor 488 or 594 (Molecular Probes, Eugene, OR) were used as secondary antibodies. .. For filipin or Nile Red staining, paraformaldehyde-fixed cells were incubated with 0.5 mg/ml filipin (Sigma, Munich, Germany) or 5 μg/ml Nile Red (Molecular Probes) in PBS for 60 min and washed with PBS. .. After filipin staining, immunofluorescence labeling was performed without a separate permeabilization step.

Article Title: Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells
Article Snippet: Paragraph title: Filipin staining ... Cells were rinsed three more times with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h on a shaker in the dark.

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Paragraph title: Filipin staining and image analysis ... Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature.

Article Title: Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation
Article Snippet: After 5 days, LPS (1 µg/mL, Sigma-Aldrich, Cat#L2630) was added and cells collected 48 h later. .. Cells were washed in 2% FBS, 2 µM EDTA PBS (MACS buffer), and then stained with filipin (0.05 mg/mL Sigma-Aldrich, Cat#F9765) at 37 °C for 1 h, CTXb (Cholera Toxin B subunit FITC conjugate—8 µg/mL, Sigma-Aldrich, Cat#C1655) or FITC- conjugated anti-human HLA-DR (BD, Cat#347363) or CD80 (BD, Cat#560926) 4 °C for 30 min in the dark. .. Following staining, cells were washed with 2% FBS, 2 µM EDTA PBS (MACS buffer) and analysed by flow cytometry.

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    Millipore filipin iii
    Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL <t>filipin</t> <t>III</t> for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p
    Filipin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Anandamide exerts its antiproliferative actions on cholangiocarcinoma by activation of the GPR55 receptor

    doi: 10.1038/labinvest.2011.62

    Figure Lengend Snippet: Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p

    Article Snippet: The inhibitors utilized were the lipid raft disruptors, methyl-β-cyclodextrin (0.1 mM), , and filipin III (1 mg/mL) ( , ), and the JNK inhibitor (10−7 M; SP600125; EMD Chemicals, Gibbstown, NJ) ( ).

    Techniques: Activation Assay